Preparation and Uses of Obeticholic Acid

ABSTRACT

The present invention relates to obeticholic acid: 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt, solvate or amino acid conjugate thereof. Obeticholic acid is useful for the treatment or prevention of a FXR mediated disease or condition, cardiovascular disease or cholestatic liver disease, and for reducing HDL cholesterol, for lowering triglycerides in a mammal, or for inhibition of fibrosis. The present invention also relates to processes for the synthesis of obeticholic acid.

RELATED APPLICATION

This application claims priority under 35 U.S.C. §119(e) to U.S.provisional application No. 61/661,531 filed on Jun. 19, 2012 which isincorporated herein by reference.

SUMMARY OF THE INVENTION

The present invention relates to obeticholic acid, an agonist for FXR,processes of preparation for obeticholic acid, pharmaceuticalformulations comprising obeticholic acid, and the therapeutic use of thesame.

The present invention relates to a crystalline obeticholic acid Form Ccharacterized by an X-ray diffraction pattern including characteristicpeaks at about 4.2, 6.4, 9.5, 12.5, and 16.7 degrees 2-Theta. Thecrystalline obeticholic acid Form C is characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 5and further characterized by a Differential Scanning calorimetry (DSC)thermogram having an endotherm value at about 98±2° C.

The present invention relates to a process for preparing obeticholicacid Form 1, comprising the step of converting crystalline obeticholicacid to obeticholic acid Form 1.

The present invention relates to a process for preparing obeticholicacid Form 1, comprising the steps of reacting3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ to formcrystalline obeticholic acid and converting crystalline obeticholic acidto obeticholic acid Form 1.

The present invention relates to a process for preparing obeticholicacid Form 1, comprising the steps of reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid;reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ toform crystalline obeticholic acid; and converting crystallineobeticholic acid to obeticholic acid Form 1.

The present invention relates to a process for preparing obeticholicacid Form 1, comprising the steps of reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl esterwith NaOH to form E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid; reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid;reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ toform crystalline obeticholic acid, and converting crystallineobeticholic acid to obeticholic acid Form 1.

The present invention relates to a process for preparing obeticholicacid Form 1, comprising the steps of reacting3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester withCH₃CHO to form E- or E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oicacid methyl ester; reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl esterwith NaOH to form E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid; reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid;reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ toform crystalline obeticholic acid, and converting crystallineobeticholic acid to obeticholic acid Form 1.

The present invention relates to a process for preparing obeticholicacid Form 1, comprising the steps of reacting3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester withLi[N(CH(CH₃)₂)₂] and Si(CH₃)₃Cl to form3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester; reacting3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester withCH₃CHO to form E- or E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oicacid methyl ester; reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl esterwith NaOH to form E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid; reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid;reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ toform crystalline obeticholic acid, and converting crystallineobeticholic acid to obeticholic acid Form 1.

The present invention relates to a process for preparing obeticholicacid Form 1, comprising the steps of reacting3α-hydroxy-7-keto-5β-cholan-24-oic acid with CH₃OH and H₂SO₄ to form3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester; reacting3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester withLi[N(CH(CH₃)₂)₂] and Si(CH₃)₃Cl to form3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester; reacting3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester withCH₃CHO to form E- or E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oicacid methyl ester; reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl esterwith NaOH to form E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid; reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid;reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ toform crystalline obeticholic acid, and converting crystallineobeticholic acid to obeticholic acid Form 1.

The present invention relates to a process for preparing obeticholicacid Form 1, wherein converting crystalline obeticholic acid Form C toobeticholic acid Form 1 comprises the step of dissolving crystallineobeticholic acid Form C in aqueous NaOH solution and adding HCl.

The present invention relates to a process for preparing obeticholicacid Form 1, wherein in reacting3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ to formcrystalline obeticholic acid is carried out at a temperature at about85° C. to about 110° C. in a basic aqueous solution.

The present invention relates to a process for preparing obeticholicacid Form 1, wherein reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid iscarried out at a temperature at about 100° C. to about 105° C. and at apressure at about 4 to about 5 bars.

The present invention relates to a process for preparing obeticholicacid Form 1, wherein reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl esterwith NaOH to form E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid is carried outat a temperature at about 20° C. to about 60° C.

The present invention relates to a process for preparing obeticholicacid Form 1, wherein reacting3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester withCH₃CHO to form E- or E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oicacid methyl ester is carried out in a polar aprotic solvent at atemperature at about −50° C. to about −70° C. in the presence of BF₃.

The present invention relates to a process for preparing obeticholicacid Form 1, wherein reacting 3α-hydroxy-7-keto-5β-cholan-24-oic acidmethyl ester with Li[N(CH(CH₃)₂)₂] and Si(CH₃)₃Cl to form3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester iscarried out in a polar aprotic solvent at a temperature at about −10° C.to about −30° C.

The present invention relates to a process for preparing obeticholicacid Form 1, wherein reacting 3α-hydroxy-7-keto-5β-cholan-24-oic acidwith CH₃OH and H₂SO₄ to form 3α-hydroxy-7-keto-5β-cholan-24-oic acidmethyl ester is heated for about 3 hours and the pH of the reactionmixture is adjusted with an aqueous basic solution to a pH-value ofabout 6.5 to about 8.0.

The present invention relates to a obeticholic acid, or apharmaceutically acceptable salt, solvate or amino acid conjugatethereof, having a potency of greater than about 98%, greater than about98.5%, greater than about 99.0%, or greater than about 99.5%. Thepresent invention relates to a pharmaceutical composition comprisingobeticholic acid Form 1 produced by a process of the invention and apharmaceutically acceptable carrier.

The present invention relates to a method of treating or preventing anFXR mediated disease or condition in a subject comprise of administeringan effective amount of obeticholic acid Form 1. The disease or conditionis selected from biliary atresia, cholestatic liver disease, chronicliver disease, nonalcoholic steatohepatitis (NASH), hepatitis Cinfection, alcoholic liver disease, primary biliary cirrhosis (PBC),liver damage due to progressive fibrosis, liver fibrosis, andcardiovascular diseases including atherosclerosis, arteriosclerosis,hypercholesteremia, and hyperlipidemia. The present invention relates toa method for lowering triglycerides in a subject comprise ofadministering an effective amount of obeticholic acid Form 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a HPLC-UV/MS chromatogram of crude compound 5 of Step 4 ofExample 1 injected at 1 mg/mL, injection volume 3 μl. The chromatogramis obtained according to the method described in Example 2.

FIG. 2 is a HPLC-UV/MS chromatogram of compound 5 of Step 4 of Example1, purified reference injected at 1 mg/mL, injection volume 20 μL. Thechromatogram is obtained according to the method described in Example 2.

FIG. 3 is a UV chromatogram of crude compound 5 of step 4 of Example 1using HPLC method. The chromatogram is obtained according to the methoddescribed in Example 2.

FIG. 4A is an accurate ion trace of m/z 850.61914±3 ppm from the mainpeak fraction (RT 29.0 min) of compound 5 of Step 4 of Example 1, purelyisolated with HPLC method (see Example 2).

FIG. 4B is an accurate ion trace of m/z 850.61914±3 ppm from the minorpeak fraction (RT 29.9 min) of compound 5 of Step 4 of Example 1, purelyisolated with HPLC method (see Example 2).

FIG. 4C is an accurate ion trace of m/z 850.61914±3 ppm from crudecompound 5 of Step 4 of Example 1 (see Example 2).

FIG. 4D is an accurate ion trace of m/z 850.61914±3 ppm from compound 5of Step 4 of Example 1, purified reference (see Example 2).

FIG. 5 is an XRPD diffractogram of crystalline obeticholic acid Form C(see Example 3).

FIG. 6 shows TGA and DSC Thermograms of crystalline obeticholic acidForm C (see Example 3).

FIG. 7 shows VT-XRPD diffractograms of crystalline obeticholic acid at25° C., 110° C., and 120° C. (see Example 3).

FIG. 8A is a GVS isotherm plot of crystalline obeticholic acid Form C(see Example 3).

FIG. 8B is a GVS kinetic plot of crystalline obeticholic acid Form C(see Example 3).

FIG. 8C shows XRPD diffractograms of crystalline obeticholic acid Form Cbefore and after GVS analysis (see Example 3).

FIG. 9 shows XRPD diffractograms of crystalline obeticholic acid Form Cbefore and after storage at 40° C./75% RH (see Example 3).

FIG. 10 is an XRPD diffractogram of batch 1 of obeticholic acid Form 1(see Example 5).

FIG. 11 shows the XRPD diffractorgraphs for batches 1, 2, 3, 4, 5 and 6of obeticholic acid Form 1 (see Example 5).

FIG. 12 is a NMR spectrum of batch 1 of obeticholic acid Form 1 ind₆-DMSO (see Example 5).

FIG. 13 shows the ¹H NMR spectra for batches 1, 2, 3, 4, 5 and 6 ofobeticholic acid Form 1 (see Example 5).

FIG. 14 is an expansion of ¹³C DEPTQ NMR spectrum of obeticholic acidForm 1 from region 10-75 ppm (see Example 5).

FIG. 15 is an expansion of ¹³C DEPT135 NMR spectrum of obeticholic acidForm 1 suppressing quaternary carbons from region 0-75 ppm (see Example5).

FIG. 16 is a quantitative ¹³C NMR of obeticholic acid Form 1 (seeExample 5).

FIG. 17 is an expanded view of peaks at 32.3 ppm of FIG. 16 (see Example5).

FIG. 18 is a FT-IR spectrum of batch 1 of obeticholic acid Form 1 (seeExample 5).

FIG. 19 shows TGA and DSC thermograms of batch 1 of obeticholic acidForm 1 (see Example 5).

FIG. 20 shows modulated DSC thermograms of batch 1 of obeticholic acidForm 1 (see Example 5).

FIG. 21 shows the TGA traces of batches 1, 2, 3, 4, 5, and 6 ofobeticholic acid Form 1 (see Example 5).

FIG. 22 shows the DSC traces of batches 1, 2, 3, 4, 5, and 6 ofobeticholic acid Form 1 (see Example 5).

FIG. 23A is a picture of batch 1 of obeticholic acid Form 1 underpolarized light microscopy. FIG. 23B is a picture of batch 2 ofobeticholic acid Form 1 under polarized light microscopy. FIG. 23C is apicture of batch 3 of obeticholic acid Form 1 under polarized lightmicroscopy. FIG. 23D is a picture of batch 4 of obeticholic acid Form 1under polarized light microscopy. FIG. 23E is a picture of batch 5 ofobeticholic acid Form 1 under polarized light microscopy. FIG. 23F is apicture of batch 6 of obeticholic acid Form 1 under polarized lightmicroscopy.

FIG. 24 shows GVS isotherm plot of batch 1 of obeticholic acid Form 1(see Example 5).

FIG. 25 shows GVS kinetics plot of batch 1 of obeticholic acid Form 1(see Example 5).

FIG. 26 shows XRPD diffractograms of batch 1 of obeticholic acid Form 1before and after GVS (see Example 5).

FIG. 27 is a graph of the measurement of pKa at three differentmethanol/water ratios for obeticholic acid Form 1 (see Example 5).

FIG. 28 is a Yasuda-Shedlovsky plot for obeticholic acid Form 1 (seeExample 5).

FIG. 29 is a graph showing the distribution of the species depending onpH for obeticholic acid Form 1 (see Example 5).

FIG. 30 is a graph showing the difference curve obtained bypotentiometry for obeticholic acid Form 1 (see Example 5).

FIG. 31 shows the lipophilicity profile of obeticholic acid Form 1 (seeExample 5).

FIG. 32 shows the XRPD diffractograms of batch 1 of obeticholic acidForm 1 after storage at 40° C./75% RH (see Example 5).

FIG. 33 shows the XRPD diffractograms of batch 1 of obeticholic acidForm 1 after storage at 25° C./97% RH (see Example 5).

FIG. 34 shows a view of the molecule of obeticholic acid Form G from thecrystal structure showing anisotropic atomic displacement ellipsoids forthe non-hydrogen atoms at the 50% probability level (see Example 6).

FIG. 35 shows a view of the intermolecular hydrogen bonds of the crystalstructure of obeticholic acid Form G where hydrogen bondings are shownin dashed lines (See Example 6).

FIG. 36 shows an XRPD overlay of the simulated powder pattern,experimental patterns of the collected crystal, and obeticholic acidForm G (see Example 6).

FIG. 37 shows a graph of the plasma obeticholic acid profile vs. timeafter oral administration of 20 mg/kg of obeticholic acid Form 1 andcrystalline Form F (see Example 7).

FIG. 38 shows a graph of the plasma concentration of tauro conjugate ofobeticholic acid Form 1 and crystalline Form F at different timeinterval after the administration (see Example 7).

FIG. 39 shows the DSC curve of Form 1 (see Example 7).

FIG. 40 shows the DSC curve of Form F (see Example 7)

DETAILED DESCRIPTION OF THE INVENTION

The present application is directed to obeticholic acid, apharmaceutically active ingredient (also known as INT-747) having thechemical structure:

including, substantially pure obeticholic acid, a process for thepreparation of obeticholic acid comprising crystalline obeticholic acidas a synthetic intermediate, and analytical methods for confirming thepresence and purity of obeticholic acid and synthetic intermediates inthe process to prepare obeticholic acid. The present application alsodescribes pharmaceutical compositions and formulations of obeticholicacid and uses for such compositions.

Process to Prepare Obeticholic Acid

The present application is directed to a process for preparing highlypure obeticholic acid. The process of the present application is shownin Scheme 1. The process is a 6-step synthesis followed by onepurification step to produce highly pure obeticholic acid.

The process of the present invention also includes a process accordingto Scheme 1 where compounds 4 and 5 are each comprised of a mixture ofthe E and Z isomers as illustrated by the structures of compounds 4A and5A below:

In one embodiment, the E/Z isomer ratio ofE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester(4A) is about 50%, greater than about 60%, greater than about 70%,greater than about 80%, greater than about 83%, greater than about 85%,greater than about 90%, greater than about 93%, greater than about 95%,or greater than about 99%. In one embodiment, the E/Z ratio isdetermined by HPLC. In one embodiment, the ratio is greater than about80%. In one embodiment, the ratio is greater than about 83%. In oneembodiment, the ratio is greater than about 85%. In one embodiment, theratio is greater than about 90%. In one embodiment, the ratio is greaterthan about 93%. In one embodiment, the ratio is greater than about 95%.In one embodiment, the ratio is greater than about 99%.

In one embodiment, the E/Z isomer ratio ofE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A) is about50%, greater than about 60%, greater than about 70%, greater than about80%, greater than about 83%, greater than about 85%, greater than about90%, greater than about 93%, greater than about 95%, or greater thanabout 99%. In one embodiment, the E/Z ratio is determined by HPLC. Inone embodiment, the ratio is greater than about 80%. In one embodiment,the ratio is greater than about 83%.

In one embodiment, the ratio is greater than about 85%. In oneembodiment, the ratio is greater than about 90%. In one embodiment, theratio is greater than about 93%. In one embodiment, the ratio is greaterthan about 95%. In one embodiment, the ratio is greater than about 99%.

The process of the present application has never been reported in theart. The process is a 6-step synthesis followed by one purificationstep. Step 1 is the esterification of the C-24 carboxylic acid of 7-ketolithocholic acid (KLCA) using methanol in the presence of acidiccatalysis and heat to produce the methyl ester compound 1. Step 2 issilylenol ether formation from compound 1 using a strong base followedby treatment with chlorosilane to produce compound 3. Step 3 is an aldolcondensation reaction of the silylenol ether compound 3 and acetaldehydeto produce compound 4 (or compound 4A). Step 4 is ester hydrolysis i.e.,saponification of the C-24 methyl ester of compound 4 (or compound 4A)to produce the carboxylic acid compound 5 (or compound 5A). Step 5 isthe hydrogenation of the 6-ethylidene moiety of compound 5 (or compound5A) followed by isomerization to produce compound 6. Step 6 is theselective reduction of the 7-keto group of compound 6 to a 7α-hydroxygroup to produce crystalline obeticholic acid. Step 7 is the conversionof crystalline obeticholic acid to obeticholic acid Form 1.

The process of the present invention relates to a process for preparingobeticholic acid Form 1, where the process utilizes a crystalline formof obeticholic acid as a synthetic intermediate.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1, comprising the step of convertingcrystalline obeticholic acid to obeticholic acid Form 1.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1, comprising the steps of

reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6) with NaBH₄to form crystalline obeticholic acid, and

converting crystalline obeticholic acid to obeticholic acid Form 1.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1, comprising the steps of

reacting E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A)with Pd/C and hydrogen gas to form3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6),

reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6) with NaBH₄to form crystalline obeticholic acid, and converting crystallineobeticholic acid to obeticholic acid Form 1.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1, comprising the steps of

reacting E-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5) withPd/C and hydrogen gas to form3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6),

reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6) with NaBH₄to form crystalline obeticholic acid, and

converting crystalline obeticholic acid to obeticholic acid Form 1.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1, comprising the steps of

reacting E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methylester (4A) with NaOH to formE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A),

reacting E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A)with Pd/C and hydrogen gas to form3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6),

reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6) with NaBH₄to form crystalline obeticholic acid, and

converting crystalline obeticholic acid to obeticholic acid Form 1.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1, comprising the steps of

reacting E-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methylester (4) with NaOH to formE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5),

reacting E-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5) withPd/C and hydrogen gas to form3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6),

reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6) with NaBH₄to form crystalline obeticholic acid, and

converting crystalline obeticholic acid to obeticholic acid Form 1.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1, comprising the steps of

reacting 3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester(3) with CH₃CHO to formE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester(4A),

reacting E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methylester (4A) with NaOH to formE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A),

reacting E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A)with Pd/C and hydrogen gas to form3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6),

reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6) with NaBH₄to form crystalline obeticholic acid, and

converting crystalline obeticholic acid to obeticholic acid Form 1.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1, comprising the steps of

reacting 3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester(3) with CH₃CHO to formE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester (4),

reacting E-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methylester (4) with NaOH to formE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5),

reacting E-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5) withPd/C and hydrogen gas to form3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6),

reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6) with NaBH₄to form crystalline obeticholic acid, and

converting crystalline obeticholic acid to obeticholic acid Form 1.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1, comprising the steps of

reacting 3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester (1) withLi[N(CH(CH₃)₂)₂] and Si(CH₃)₃Cl to form3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester (3),

reacting 3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester(3) with CH₃CHO to formE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester(4A),

reacting E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methylester (4A) with NaOH to formE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A),

reacting E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A)with Pd/C and hydrogen gas to form3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6),

reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6) with NaBH₄to form crystalline obeticholic acid, and

converting crystalline obeticholic acid to obeticholic acid Form 1.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1, comprising the steps of

reacting 3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester (1) withLi[N(CH(CH₃)₂)₂] and Si(CH₃)₃Cl to form3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester (3),

reacting 3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester(3) with CH₃CHO to formE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester (4),

reacting E-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methylester (4) with NaOH to formE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5),

reacting E-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5) withPd/C and hydrogen gas to form3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6),

reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6) with NaBH₄to form crystalline obeticholic acid, and

converting crystalline obeticholic acid to obeticholic acid Form 1.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1, comprising the steps of

reacting 3α-hydroxy-7-keto-5β-cholan-24-oic acid (KLCA) with CH₃OH andH₂SO₄ to form 3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester (1).

reacting 3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester (1) withLi[N(CH(CH₃)₂)₂] and Si(CH₃)₃Cl to form3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester (3),

reacting 3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester(3) with CH₃CHO to formE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester(4A),

reacting E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methylester (4A) with NaOH to formE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A),

reacting E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A)with Pd/C and hydrogen gas to form3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6),

reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6) with NaBH₄to form crystalline obeticholic acid, and

converting crystalline obeticholic acid to obeticholic acid Form 1.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1, comprising the steps of

reacting 3α-hydroxy-7-keto-5β-cholan-24-oic acid (KLCA) with CH₃OH andH₂SO₄ to form 3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester (1).

reacting 3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester (1) withLi[N(CH(CH₃)₂)₂] and Si(CH₃)₃Cl to form3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester (3),

reacting 3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester(3) with CH₃CHO to formE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester (4),

reacting E-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methylester (4) with NaOH to formE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5),

reacting E-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5) withPd/C and hydrogen gas to form3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6),

reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6) with NaBH₄to form crystalline obeticholic acid, and

converting crystalline obeticholic acid to obeticholic acid Form 1.

In one embodiment, the present invention relates to a process forpreparing obeticholic acid Form 1 using crystalline obeticholic acid. Inanother embodiment, the crystalline obeticholic acid is Form C. In oneembodiment, the crystalline obeticholic acid Form C is characterized byan X-ray diffraction pattern similar to that set forth in FIG. 5. In oneembodiment, the crystalline obeticholic acid Form C is crystallized andrecrystallized from n-butyl acetate.

Step 1

Step 1 is the reaction of 3α-hydroxy-7-keto-5β-cholan-24-oic acid (KLCA)with CH₃OH and H₂SO₄ to form 3α-hydroxy-7-keto-5β-cholan-24-oic acidmethyl ester (1). In one embodiment of step 1, the reaction mixture isheated for about 3 hours and the pH of the reaction mixture is adjustedwith an aqueous basic solution to a pH-value of about 6.5 to about 8.0.In one embodiment, the isolation of 3α-hydroxy-7-keto-5β-cholan-24-oicacid methyl ester (1) further comprises treatment with activated carbon.In one embodiment, the isolation of 3α-hydroxy-7-keto-5β-cholan-24-oicacid methyl ester (1) does not further comprise treatment with activatedcarbon. In one embodiment, isolation of3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester (1) without thetreatment with activated carbon affords a higher yield. In oneembodiment, reacting 3α-hydroxy-7-keto-5β-cholan-24-oic acid (1) withCH₃OH and H₂SO₄ is carried out in methanol. In one embodiment, the basicsolution is an aqueous NaOH solution. In one embodiment, the pH-value isabout 7.0 to about 7.5.

In one embodiment, the methyl alcohol acts as the methylating reagent aswell as the reaction solvent. In one embodiment, the solution containingthe product is treated with activated carbon for about 30 minutes andfiltered to remove the carbon solids. In one embodiment, the solutioncontaining the product is not treated with activated carbon. Toprecipitate the product, water at about 5° C. to about 20° C. andseeding material are added. In another embodiment, the water is at about10° C. to about 15° C. In one embodiment, the product is isolated with acentrifuge and washed with a mixture of methanol and water. In oneembodiment, the water content of the wet material is quantified by KarlFischer (KF). In one embodiment, the material is dried in a tumble dryerbefore use in the next step. In one embodiment, the material is notdried before use in the next step.

Step 2

Step 2 is the reaction of 3α-hydroxy-7-keto-5β-cholan-24-oic acid methylester (1) with Li[N(CH(CH₃)₂)₂] and Si(CH₃)₃Cl to form3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester (3). Inone embodiment, step 2 is carried out in a polar aprotic solvent at atemperature at about −10° C. to about −30° C. In one embodiment, thepolar aprotic solvent is tetrahydrofuran. In one embodiment, thetemperature is about −20° C. to about −25° C. In one embodiment,reacting 3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester (1) withLi[N(CH(CH₃)₂)₂] and Si(CH₃)₃Cl is stirred for about 2 hours.

In one embodiment, compound 1 is charged into the reactor under inertconditions. In another embodiment, residual water and methanol areremoved by repeated azeotropic distillation at about 65° C. and normalpressure. In another embodiment, THF is added to the residue asnecessary and the distillation is repeated about 4 times. In anotherembodiment, the distillation is repeated about 3 times, about 2 times,or about 1 time. In one embodiment, the remaining solution containingthe product has a final water content of ≦0.05% (Karl FischerTitration). Water can hydrolyze chlorotrimethylsilane, which is addedlater in this step. In one embodiment, the solution of the product ispre-cooled to about −10° C. to about

−30° C. and then chlorotrimethylsilane is added. In another embodiment,the solution is pre-cooled to about −20° C. to about −25° C. In oneembodiment, a strong base and THF are charged to a separate reactor andcooled to about −10° C. to about −30° C. In one embodiment, the strongbase is lithium diisopropylamide. In another embodiment, the reactor isinert, e.g., under a nitrogen or argon atmosphere. In anotherembodiment, the solution of base and THF is cooled to about −20° C. toabout −25° C. In one embodiment, the dry, cooled solution of3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester, THF, andchlorotrimethylsilane is charged into the basic solution at about −10°C. to about −30° C. In another embodiment, the temperature is about −20°C. to about −25° C. In one embodiment, the reaction mixture is stirredfor about 2 hours. In one embodiment, for the workup, the reactionmixture is added to a pre-cooled acidic solution. In another embodiment,the acidic solution is an aqueous citric acid solution. In oneembodiment, after the addition, the aqueous phase is separated anddiscarded. In one embodiment, the solvent is removed from the organicphase, by vacuum distillation at about 50° C. In one embodiment, theisolated residue is 3α,7α-ditrimethylsilyloxy-5β-chol-6-en-24-oic acidmethyl ester (3) is used ‘as is’ in the next step. Alternatively,compound 3 can be purified before Step 3.

Step 3

Step 3 is the reaction of 3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oicacid methyl ester (3) with CH₃CHO to form3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester (4).In one embodiment, step 3 is carried out in a polar aprotic solvent at atemperature at about −50° C. to about −70° C. in the presence of BF₃. Inone embodiment, the polar aprotic solvent is dichloromethane. In oneembodiment, the BF₃ is a 16% wt. solution in acetonitrile. In oneembodiment, the temperature is about −60° C. to about −65° C.

In one embodiment, compound 3 in a polar aprotic solvent is charged intoan inert reactor. In another embodiment, the polar aprotic solvent isthe residual solvent from the previous step (e.g., THF). In oneembodiment, THF is added to help distill off residual water anddiisopropylamine. At a maximum temperature of about 50° C., residualamounts of the polar aprotic solvent are distilled off under vacuum. Thewater content in the residue containing compound 3 is limited to <0.5%(Karl Fischer titration). The residue containing compound 3 is thendissolved in a polar aprotic solvent and pre-cooled to about −50° C. toabout −70° C. The polar aprotic solvent is dichloromethane. In anotherembodiment, residue containing compound 3 in the polar aprotic solventis pre-cooled to about −60° C. to about −65° C. Acetaldehyde (CH₃CHO) isadded. A polar aprotic solvent and boron trifluoride (BF₃) solvatedcomplex are charged into a separate reactor and then cooled to about−50° C. to about −70° C. In another embodiment, the polar aproticsolvent is dichloromethane. In another embodiment, the boron trifluoridesolvated complex is a boron trifluoride acetonitrile complex. Thetemperature of the BF₃ solution is about −60° C. to about −65° C. Thesolution containing compound 3 and acetaldehyde is added to the BF₃solution at about −60° C. to about −65° C. In another embodiment, thesolution containing compound 3 and acetaldehyde is dry. In oneembodiment, the reaction mixture is stirred for about two hours at about−60° C. to about −65° C., heated up to about 23° C. to about 28° C.,stirred for another about 2 hours and cooled to about 2° C. to about 10°C. for the hydrolysis/work-up. In one embodiment, the total time foraddition and stirring is about 4 hours. In one embodiment, for theworkup, the cooled solution from the reactor is added to a pre-cooledaqueous basic solution. In another embodiment, the aqueous basicsolution is about 50% wt. sodium hydroxide (NaOH; caustic soda). In oneembodiment, the phases are separated and the (lower) organic layer istransferred to a separate reactor. In one embodiment, from the organiclayer, the solvent is removed by distillation at not more than (NMT) 50°C. as far as possible. In one embodiment, the residue comprises3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester (4)and some remaining acetonitrile and dichloromethane. It is understoodthat step 4 may form E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oicacid methyl ester (4A). The product of Step 3 is taken on directly toStep 4.

Step 4

Step 4 is the reaction of3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester (4)with NaOH to form E-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid(5). In one embodiment, prior to step 4, the residue from step 3 isheated to about 45° C. to about 60° C. to remove residual amounts ofsolvent. In one embodiment, the temperature is about 49° C. to about 55°C. In one embodiment, the ester hydrolysis reaction reacting3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester (4)with NaOH is carried out at about 20° C. to about 25° C. in methanol,water, and a NaOH solution.

In one embodiment, reacting3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester (4) ischarged into a reactor. In another embodiment, the reactor is inert,e.g., under a nitrogen or argon atmosphere. At a temperature of NMT 50°C., residual amounts of solvent are distilled off under vacuum. In oneembodiment, the residue is heated up to about 45° C. to about 60° C. Inanother embodiment, the residue is heated up to about 49° C. to about55° C. In another embodiment, the residue from Step 3 (compound 4) isdissolved in methanol and water and an aqueous basic solution. Inanother embodiment, the aqueous basic solution is about 50% wt. sodiumhydroxide (NaOH; caustic soda). The ester hydrolysis reaction of Step 4is carried out at about 20° C. to about 60° C. and stirred until thehydrolysis reaction is complete. In one embodiment, the ester hydrolysisis carried out at about 20° C. to about 25° C. The pH of the reactionmixture is checked to verify it is >12. If the pH is <12, thenadditional NaOH is added. The reaction mixture is diluted with water andthe temperature is adjusted to about 20° C. to about 35° C. In anotheraspect, the reaction mixture is diluted with water and the temperatureis adjusted to about 25° C. to about 35° C. In one embodiment, for theworkup, the phases are separated and the lower aqueous layer istransferred into a separate reactor and the organic layer is discarded.Compound 5 is in the aqueous phase. In one embodiment, ethyl acetate andan acid were added to the aqueous phase containing compound 5 withintensive stirring to the aqueous layer. In another embodiment, the acidis an aqueous citric acid solution. In one embodiment, the phases areseparated and the lower aqueous layer is discarded. Compound 5 is in theorganic layer. In one embodiment, ethyl acetate is distilled off fromthe organic layer and replaced with ethyl acetate. In one embodiment,the distillation is repeated until the water content of the distillateis NMT 1% or until a constant boiling point is reached. In oneembodiment, the suspension is cooled to about 10° C. to about 30° C. andisolated and washed with ethyl acetate. In another embodiment, theresulting suspension containing compound 5 is cooled to about 20° C. toabout 25° C. In one embodiment, drying of the resulting product is doneunder vacuum (e.g, tumble dryer) at about 60° C.

In one embodiment, crudeE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5) iscrystallized using ethanol. In one embodiment, ethanol and crudecompound 5 are charged into reactor. In another embodiment, the reactoris inert. In one embodiment, to dissolve the crude compound 5, themixture is heated to reflux. In one embodiment, mixture is cooled in acontrolled cooling ramp to about 15° C. to about 20° C. In oneembodiment, the crystalline compound 5 is isolated using a centrifugeand then washed with ethyl acetate. In one embodiment, drying ofcrystalline compound 5 is done under vacuum (e.g, tumble dryer) and atabout 60° C. A sample can be taken to measure assay, purity, andmoisture of the purified compound 5. In one embodiment, purifiedcompound 5 contains both E and Z isomers of3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid. In one embodiment,the E to Z ratio is about 99:1, about 98:2, about 95:5, about 90:10,about 85:15, about 80:20, about 75:25, about 70:30, about 65:35, about60:40, about 55:45, or about 50:50. See Example 2 for full detailsregarding the identification and characterization of purified compound5.

Step 4 can also be carried out starting with a compound that is amixture of E/Z isomer. For example, Step 4 is the reaction ofE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester(4A) with NaOH to formE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A). In oneembodiment, prior to step 4, the residue from step 3 is heated about 45°C. to about 60° C. to remove residual amounts of solvent. In oneembodiment, the temperature is about 49° C. to about 55° C. In oneembodiment, the ester hydrolysis reaction involving reactingE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester(4A) with NaOH is carried out at about 20° C. to about 25° C. inmethanol, water, and a NaOH solution. In one embodiment, the NaOHsolution is a 50% wt. aqueous solution.

In one embodiment, reactingE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl ester(4A) is charged into a reactor. In another embodiment, the reactor isinert, e.g., under a nitrogen or argon atmosphere. At a temperature ofNMT 50° C., residual amounts of solvent are distilled off under vacuum.In one embodiment, the residue is heated up to about 45° C. to about 60°C. In one embodiment, the temperature is about 49° C. to about 55° C. Inone embodiment, the residue from step 3 (compound 4A) is dissolved inmethanol and water and an aqueous basic solution. In another embodiment,the aqueous basic solution is about 50% wt. sodium hydroxide (NaOH;caustic soda). The ester hydrolysis reaction of step 4 is carried out atabout 20° C. to about 60° C. and stirred until the hydrolysis reactionis complete. In one embodiment, the ester hydrolysis is carried out atabout 20° C. to about 25° C. The pH of the reaction mixture is checkedto verify it is >12. If the pH is <12, then additional NaOH is added.The reaction mixture is diluted with water and the temperature isadjusted to about 25° C. to about 35° C. In one embodiment, for theworkup, the phases are separated and the lower aqueous layer istransferred into a separate reactor and the organic layer is discarded.Compound 5A is in the aqueous phase. In one embodiment, ethyl acetateand an acid were added to the aqueous phase containing compound 5A withintensive stirring to the aqueous layer. In another embodiment, the acidis an aqueous citric acid solution. In one embodiment, the phases areseparated and the lower aqueous layer is discarded. Compound 5A is inthe organic layer. In one embodiment, ethyl acetate is distilled offfrom the organic layer and replaced with ethyl acetate. In oneembodiment, the distillation is repeated until the water content of thedistillate is NMT 1% or until a constant boiling point is reached. Inone embodiment, the suspension is cooled to about 10° C. to about 30° C.and isolated and washed with ethyl acetate. In another embodiment, theresulting suspension containing compound 5A is cooled to about 20° C. toabout 25° C. In one embodiment, drying of the resulting product is doneunder vacuum (e.g, tumble dryer) at about 60° C. Compound 5A can becarried on without purification to Step 5.

In one embodiment, crudeE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A) iscrystallized using ethanol. In one embodiment, ethanol and crudecompound 5A are charged into reactor. In another embodiment, the reactoris inert. In one embodiment, to dissolve the crude compound 5A, themixture is heated to reflux. In one embodiment, mixture is cooled in acontrolled cooling ramp to about 15° C. to about 20° C. In oneembodiment, the crystalline compound 5A is isolated using a centrifugeand then washed with ethyl acetate. In one embodiment, drying ofcrystalline compound 5A is done under vacuum (e.g, tumble dryer) and atabout 60° C. In one embodiment, the isolated crystalline product of step4 is compound 5.

Alternative Step 4

Compound 5 can be prepared according to an alternative method. In oneembodiment, compound 4 is charged into the inert reactor. At about 50°C. (maximum) residual amounts of solvent (e.g., acetonitrile,dichloromethane) may be distilled off under vacuum. The residue isdissolved in methanol and cooled. Tap-water and caustic soda (50% weightNaOH) are added. In one embodiment, the reaction mixture is stirred forabout four hours at about 20° C. to about 25° C. The solution is dilutedwith tap-water and toluene is added. After stirring, the phases areseparated and the lower, aqueous layer is transferred into the inertreactor. The organic layer is discarded. Acetic acid ethylester and asolution of citric acid are added during intensive stirring to theaqueous layer. The phases are separated and the lower, aqueous layer isdiscarded. The organic layer is transferred into the inert reactor. Fromthe organic layer acetic acid ethylester is distilled off and replacedwith acetic acid ethyl ester. In one embodiment, this operation isrepeated until the water content of the distillate is not more thanabout 1% or until a constant boiling point is reached. The presentsuspension is cooled to about 20° C. to about 25° C., and compound 5 isisolated and washed with acetic acid ethylester with the inertcentrifuge. Drying is done in the tumble dryer under vacuum andapproximately 60° C.

This alternative Step 4 can also be carried out starting with a compoundthat is a mixture of E/Z isomer. In one embodiment, compound 4A ischarged into the inert reactor. At about 50° C. (maximum) residualamounts of solvent (e.g., acetonitrile, dichloromethane) may bedistilled off under vacuum. The residue is dissolved in methanol andcooled. Tap-water and caustic soda (50% wt, NaOII) are added. In oneembodiment, the reaction mixture is stirred for approximately four hoursat about 20° C. to about 25° C. The solution is diluted with tap-waterand toluene is added. After stirring, the phases are separated and thelower, aqueous layer is transferred into the inert reactor. The organiclayer is discarded. Acetic acid ethylester and a solution of citric acidare added during intensive stirring to the aqueous layer. The phases areseparated and the lower, aqueous layer is discarded. The organic layeris transferred into the inert reactor. From the organic layer aceticacid ethylester is distilled off and replaced with acetic acidethylester. In one embodiment, this operation is repeated until thewater content of the distillate is not more than about 1% or until aconstant boiling point is reached. The present suspension is cooled to20° C. to 25° C., and compound 5A is isolated and washed with aceticacid ethylester with the inert centrifuge. Drying is done in the tumbledryer under vacuum and approximately 60° C.

Step 5

Step 5 is the reaction ofE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5) with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid(6). Step 5 can be carried out in one phase (hydrogenation andisomerization together) or in two phases (hydrogenation followed byisomerization). In one embodiment, Step 5 is carried out at atemperature at about 90° C. to about 110° C. and at a pressure at about4 to about 5 bars. In one embodiment, during workup, the organic phaseof the reaction mixture is treated with activated carbon. In oneembodiment, the pressure is about 4.5 to about 5.5 bars. In anotherembodiment, the pressure is about 5 bars. In one embodiment, thehydrogenation reaction mixture is allowed to stir for about 1 hour. Inone embodiment, reactingE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5) with Pd/C andhydrogen gas is heated to about 100° C. and stirred for about 2 hour toabout 5 hours. In one embodiment, reactingE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5) with Pd/C andhydrogen gas is heated to about 100° C. and stirred for about 3 hours.

In one embodiment, reactingE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5) with Pd/C andhydrogen gas is carried out in the presence of a basic solution. In oneembodiment, the basic solution is a 50% wt. sodium hydroxide (NaOH;caustic soda) solution. After the hydrogenation reaction, the reactionmixture is heated up to about 100° C. (to carry out the isomerisation ofthe C-6 position from beta configuration to alpha configuration) andthen cooled to about 40° C. to about 50° C. For the workup, the Pd/C isfiltered off. In one embodiment, to the filtrate, n-butyl acetate and anacid are added. In another embodiment, the acid is hydrochloric acid(HCl). The aqueous phase is separated and discarded after checking thepH-value to make sure that it was acidic. The organic phase containingthe product is treated with activated carbon. In one embodiment, theactivated carbon is filtered off and the resulting filtrate containingthe product is condensed by distillation and the resulting suspension iscooled to about 10° C. to about 30° C. In another embodiment, thesuspension is cooled to about 15° C. to about 20° C. The suspensioncontaining compound 6 is isolated and washed with n-butyl acetate.Compound 6 is filtered using a pressure filter. In one embodiment,drying is done in the pressure filter under vacuum at about 80° C.

In one embodiment in Step 5,E-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5), water, NaOHsolution (e.g. 50% wt.), and Pd/C are mixed at about 5 bar of H₂ gas andat a temperature at about 100° C. to about 105° C. until H₂ uptake hasceased. The reaction mixture is cooled to about 40° C. to about 50° C.and Pd/C is filtered off. Then n-butyl acetate and HCl are added to thesolution containing compound 6. In one embodiment, the aqueous phase isseparated and discarded. The organic phase containing compound 6 istreated with activated carbon. The carbon is filtered off and thefiltrate is moved to another reactor where it is reduced down bydistillation, and then the suspension is cooled to about 5° C. to about20° C. In one embodiment, compound 6 is isolated via filtration and thefiltrate is dried on the pressure filter under vacuum at about 80° C.

Step 5 can also be carried out starting with a compound that is amixture of E/Z isomer. For example, Step 5 is the reaction ofE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A) with Pd/Cand hydrogen gas and heat to form3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid (6). Step 5 can becarried out in one phase (hydrogenation and isomerization together) orin two phases (hydrogenation, followed by isomerization). In one aspect,step 5 is carried out at a temperature at about 90° C. to about 110° C.and at a pressure at about 4 to about 5 bars. In one embodiment, duringworkup, the organic phase of the reaction mixture is treated withactivated carbon. In one embodiment, the pressure is about 4.5 to about5.5 bars. In another embodiment, the pressure is about 5 bars. In oneembodiment, the hydrogenation reaction mixture is allowed to stir forabout 1 hour. In one embodiment, reactingE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A) with Pd/Cand hydrogen gas is heated to about 100° C. and stirred for about 2 hourto about 5 hours. In one embodiment, reactingE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A) with Pd/Cand hydrogen gas is heated to about 100° C. and stirred for about 3hours.

In one embodiment, reactingE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A) with Pd/Cand hydrogen gas is carried out in the presence of a basic solution. Inone embodiment, the basic solution is a 50% wt. sodium hydroxide (NaOH;caustic soda) solution. After the hydrogenation reaction, the reactionmixture is heated up to about 100° C. (to carry out the isomerisation ofthe C-6 position from beta configuration to alpha configuration) andthen cooled to about 40° C. to about 50° C. For the workup, the Pd/C isfiltered off. In one embodiment, to the filtrate, n-butyl acetate and anacid are added. In another embodiment, the acid is hydrochloric acid(HCl). The aqueous phase is separated and discarded after checking thepH-value to make sure that it was acidic. The organic phase containingthe product is treated with activated carbon. In one embodiment, theactivated carbon is filtered off and the resulting filtrate containingthe product is condensed by distillation and the resulting suspension iscooled to about 10° C. to about 30° C. In another embodiment, thesuspension is cooled to about 15° C. to about 20° C. The suspensioncontaining compound 6 is isolated and washed with n-butyl acetate.Compound 6 is filtered using a pressure filter. In one embodiment,drying is done in the pressure filter under vacuum at about 80° C.

In one embodiment in Step 5,E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5A), water,NaOH solution (e.g. 50% wt.), and Pd/C are mixed at about 5 bar of H₂gas and at a temperature at about 100° C. to about 105° C. until H₂uptake has ceased. The reaction mixture is cooled to about 40° C. toabout 50° C. and Pd/C is filtered off. Then n-butyl acetate and HCl areadded to the solution containing compound 6. In one embodiment, theaqueous phase is separated and discarded. The organic phase containingcompound 6 is treated with activated carbon. The carbon is filtered offand the filtrate is moved to another reactor where it is reduced down bydistillation, and then the suspension is cooled to about 5° C. to about20° C. In one embodiment, compound 6 is isolated via filtration and thefiltrate is dried on the pressure filter under vacuum at about 80° C.

In another embodiment, the hydrogenation/isomerization reactionsdescribed above to prepare compound 6 are carried out in two phases(starting from compound 5 or compound 5A). First, the hydrogenation iscarried out at about 4 to 5 bars and then second, the reaction mixtureis heated to about 20° C. to about 40° C. Heating the reaction mixtureisomerizes the ethyl group at the 6-position to the desired alphaconfiguration. The reaction mixture is heated until the isomerization iscomplete.

Step 6

Step 6 is the reaction of 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oicacid (6) with NaBH₄ to form crystalline obeticholic acid. In oneembodiment, Step 6 is carried out at a temperature at about 85° C. toabout 110° C. in a basic aqueous solution. In one embodiment, thetemperature is about 90° C. to about 95° C. In one embodiment, the basicaqueous solution is an aqueous NaOH solution. In one embodiment, thebasic aqueous solution is a mixture of 50% wt. NaOH solution and water.In one embodiment, the reaction mixture of compound 6 and NaBH₄ wasstirred for about 3 hours to about 5 hours. In another embodiment, thereaction mixture was stirred for about 4 hours.

For the workup, after the reaction is complete, the mixture is cooled toabout 80° C. and transferred to a cooled reactor. In one embodiment, atabout 20° C. to about 60° C., n-butyl acetate and an acid are added. Inone embodiment, the temperature is about 40° C. to about 45° C. Inanother embodiment, the acid is citric acid. The aqueous phase isseparated and discarded after checking the pH-value to make sure that itwas acidic. The organic phase containing the product is concentrated bydistillation. In one embodiment, n-butyl acetate is added to the residueand distilled off again. In one embodiment, n-butyl acetate is addedagain to the residue and then is slowly cooled down. In anotherembodiment the residue is seeded at about 50° C. In another embodiment,after crystallization has occurred, the mixture is heated to 52° C. andthen slowly cooled down to about 15° C. to about 20° C. In anotherembodiment, the residue is cooled to about 15° C. to about 20° C. In oneembodiment, the resulting obeticholic acid is washed with n-butylacetate. In one embodiment, the obeticholic acid is isolated and washedwith n-butyl acetate (e.g, in a pressure filter). In another embodiment,the pressure filter is inert. The crystalline product is dried undervacuum at about 60° C. In one embodiment, the resulting crystallineobeticholic acid is isolated from organic solvent (e.g., heptane). Seeexample 3 for full details regarding the identification andcharacterization of crystalline obeticholic acid Form C.

Step 7

Step 7 is the conversion of crystalline obeticholic acid Form C toobeticholic acid Form 1. In one embodiment, Step 7 comprises the step ofdissolving crystalline obeticholic acid Form C in aqueous NaOH solutionand adding HCl.

In one embodiment, crystalline obeticholic acid is dissolved in waterand caustic soda solution (50% wt.) at about 20° C. to about 50° C. Inone embodiment, the temperature is about 30° C. to about 40° C. In oneembodiment, the crystalline obeticholic acid is Form C. In oneembodiment, the resulting solution of crystalline obeticholic acid FormC is added to diluted acid at about 20° C. to about 50° C. In anotherembodiment, the temperature is about 30° C. to about 40° C. In anotherembodiment, the acid is hydrochloric acid (e.g., 37%). In oneembodiment, the 37% hydrochloric acid solution is diluted with water toless than about 1% by volume. In one embodiment, the 37% hydrochloricacid solution is diluted with water to about 0.7% by volume. In oneembodiment, the suspension of product in the diluted acid is stirred forabout 30 minutes at about 20° C. to about 50° C. In another embodiment,the temperature is about 30° C. to about 40° C. In one embodiment,obeticholic acid Form 1 is isolated and washed with water (e.g., in thepressure filter) at NMT about 20° C. In one embodiment, obeticholic acidForm 1 is isolated and washed with water (e.g., in the pressure filter)at NMT about 20° C. In another embodiment, the pressure filter is inert.The product is dried on the pressure filter under vacuum at atemperature of NMT about 50° C.

The process of the present application utilizes a crystallineintermediate in the preparation of obeticholic acid Form 1, whichunexpectedly led to significant improvements in the overall preparationand purity of the final product. Specifically, Step 6 of the synthesisproduces a novel crystalline form of obeticholic acid. The production ofthis crystalline form leads to substantially pure obeticholic acid Form1.

The process of the present application is an improvement over theprocesses disclosed in the prior art. The preparation of obeticholicacid is disclosed in U.S. Publication No. 2009/0062526 A1 (hereinreferred to as the “'526 publication”), U.S. Pat. No. 7,138,390(referred to herein as the “'390 patent”), and WO 2006/122977 (referredto herein as the “'977 application”).

The process to prepare obeticholic acid in the '390 patent (referred toherein as the “390 process”) is depicted in Scheme 3 (R is ethyl):

Even though this process comprises a few steps, it presents a series ofdrawbacks. In all of the steps, the reaction products are purified on achromatographic column, namely a very expensive separation method thatcannot be used on an industrial scale. Moreover, the reaction yield instep 2 is extremely low (12-13%) with a considerable decrease in theglobal yield, which is lower than 3.5%. This process also useshexamethylenphosphonamide as reactant, which is a known carcinogenicagent.

The process to prepare obeticholic acid in the '977 application isdepicted in Scheme 4.

The '977 process to prepare obeticholic acid is an 8-step syntheticprocess which includes one purification step (step 7) followed by 2additional purification steps. There are a significant number ofdifferences between the '977 process and the process of the presentapplication. Table A below describes at least some of the differencesbetween the two processes:

TABLE A Differences Between ′977 Process and Process of the ApplicationChanges Synthetic Step ′977 Process Process of the applicationAdvantages of the Change Step 1 Methanesulfonic acid Sulfuric acid Scaleand safety (mesylate) 30% ammonia (aqueous) NaOH (aqueous) Scale-up NoUse of activated carbon Improve purity/color purification/treatmenttreatment Step 2 Triethylamine Lithium diisopropylamide LDA is asuitable (Process of application (LDA) alternative reagent for this step2 combines ′977 step Process Steps 2 and 3) Toluene Tetrahydrofuran(THF) THF is a suitable alternative reagent for this step No acidicquench Quench into citric acid Scale-up solution Step 3 Borontrifluoride diethyl Boron trifluoride Safety concerns of (Process ofapplication etherate acetonitrile complex handling etherate step 3 sameas ′977 (explosion hazard with Step 4) ether) Step 4 Toluene MethanolSafety (toluene); scale (Process of application Phosphoric acid Citricacid (aqueous) Scale-up step 4 same as ′977 (aqueous) quench quench Step5) No Crystallization step is part Improve purity purification/treatmentof workup Step 5 Phosphoric acid Hydrochloric acid Scale-up (Process ofapplication (aqueous) quench (aqueous) quench step 5 combines ′977 NoUse of activated carbon Improve purity/color Process steps 6 and 7)purification/treatment treatment Purification carried outCrystallization step is part Scale-up as Step 7 of workup Step 6Dichloromethane n-Butylacetate Safety (dichloromethane) (Process ofapplication Phosphoric acid Citric acid (aqueous) Scale-up step 6combines ′977 (aqueous) quench quench Process steps 8 and 9)Purification carried out Crystallization step is part Scale and safetyas Step 9 - using of workup - using n- (dichloromethane)dichloromethane/ethyl butylacetate acetate Step 7 Ammonia solution NaOHsolution Scale-up (Process of application Phosphoric acid Hydrochloricacid Scale-up step 7 same as ′977 (aqueous) quench (aqueous) quench step10)

The differences in the process of the present application as compared tothe '977 process result in significant improvements to the process,including improvements related to scale-up optimization, safety, as wellas purity and improvements in the overall process. The purity ofobeticholic acid produced by the processes of the present application issubstantially pure. Specifically, obeticholic acid produced by theprocesses of the present application is substantially more pure thanobeticholic acid produced by processes in the prior art, including the'390 process and the '977 process. For example, a comparison of theresults presented in the Certificate of Analysis of obeticholic acidproduced by a process of the present application and obeticholic acidproduced by the '977 process are shown in the Table B below. Thepercentages of impurities were determined using HPLC methods.

TABLE B Comparison of Impurities of Obeticholic Acid Generated fromProcess of the Application and ′977 Process Specification Process of theParameter limit application ′977 process Water (KF) NMT 4.5%  1.0%  2.1%Impurity 1 and Impurity 4 NMT 0.15% <0.05% <0.05%  Impurity 2 NMT 0.15%<0.05% <0.1% Impurity 3 NMT 0.15% <0.05% <0.1% Impurity 5 NMT 3.0%  0.2% 1.0% Impurity 6 NMT 0.15% <0.05% <0.05%  Impurity 1 is6-ethylursodeoxycholic acid. Impurity 2 is3α-hydroxy-6α-ethyl-7-cheto-5β-cholan-24-oic acid. Impurity 3 is6β-ethylchenodeoxycholic acid. Impurity 4 is3α,7α-dihydroxy-6-ethyliden-5β-cholan-24-oic acid. Impurity 5 ischenodeoxycholic acid. Impurity 6 is3α(3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oyloxy)-7α-hydroxy-6α-ethyl-5β-cholan-24-oicacid (6ECDCA dimer). NMT refers to “not more than”.

Crystalline Obeticholic Acid as a Synthetic Intermediate

Obeticholic acid is currently being developed as an activepharmaceutical ingredient as a non-crystalline solid. In order tofacilitate the development of obeticholic acid, an initialcrystallization and polymorphism study was carried out in order todetermine if crystalline forms were accessible and if so, if they weresuitable for development. After a preliminary solubility screen designedto give a better understanding of the behavior of the material invarious solvents, it appeared that the material had a tendency to formgels and could possibly be crystallized. An extensive polymorph screenwas then carried out, exposing the material to a large range of solventsand crystallization conditions in order to identify and characterize asmany relevant polymorphs as possible. Five different solid forms werefound during this screen.

Three forms (A, C, and D) of obeticholic acid were mixedhydrates/solvates containing 0.25 mol eq of water and varying amounts ofa range of organic solvents. On heating, these solids lost crystallinityand the solvent at the same time and unfortunately, these solvated formswere not suitable for further development as a pharmaceutical ingredientdue to their low melting temperatures and high solvent content. It isalso noted that similar “unsuitable” forms of this type exist. Forexample, a low-melting solvated form was found in later experiments, aswell as single crystals of another form, which was shown to be amonohydrate/anisole solvate by SCXRD (Single crystal X-ray diffraction).

The two remaining forms were higher melting and potentially morepromising, but one of them (Form G) could not be reproduced on scale-up,nor repeated despite many attempts. The difficulty in producing thisform alone makes it unsuitable for development. The remainingnon-solvated Form F was reproducibly prepared, but it required extensiverecrystallization procedures and the use of nitromethane, which is atoxic solvent and may detonate if sensitized by amines, alkalis, strongacids, or high temperatures or adiabatic compression. Concerns about theresidual levels of nitromethane deemed Form F also to be unsuitable fordevelopment.

The overall results of the initial crystallization and polymorph studyrevealed that the material could form various forms of crystallinematerials, but none of the crystalline materials or forms wereconsidered suitable for development.

It was not until much later that it was discovered the importance ofproducing crystalline obeticholic acid as an intermediate in thepenultimate step of the process of the present application. Crystallineobeticholic acid could readily be isolated on large scale using theprocess of the application. This crystalline obeticholic acid wasdetermined to be consistent with Form C from the initial crystallizationand polymorph study. The formation, ease of isolation, and highly purecrystalline obeticholic acid produced as a synthetic intermediate instep 7 in the process of the present application is indeed critical tothe preparation of substantially pure obeticholic acid.

In one embodiment, the present invention relates to a crystallineobeticholic acid Form C characterized by an X-ray diffraction patternincluding characteristic peaks at about 4.2, 6.4, 9.5, 12.5, and 16.7degrees 2-Theta. In one embodiment, the X-ray diffraction patternincludes characteristic peaks at about 4.2, 6.4, 9.5, 12.5, 12.6, 15.5,15.8, 16.0, 16.7 and 19.0 degrees 2-Theta. In one embodiment, the X-raydiffraction pattern includes characteristic peaks at about 4.2, 6.4,8.3, 9.5, 11.1, 12.2, 12.5, 12.6, 15.5, 15.8, 16.0, 16.3, 16.7, 18.6 and19.0 degrees 2-Theta. In one embodiment, the X-ray diffraction patternincludes characteristic peaks at about 4.2, 6.4, 8.3, 9.5, 11.1, 12.2,12.5, 12.6, 15.5, 15.8, 16.0, 16.3, 16.7, 17.0, 17.8, 18.6, 18.8, 19.0,20.5 and 20.9 degrees 2-Theta. In one embodiment, the present inventionrelates to a crystalline obeticholic acid Form C characterized by anX-ray diffraction pattern substantially similar to that set forth inFIG. 5. In one embodiment, the X-ray diffraction pattern is collected ona diffractometer using Cu Kα radiation (40 kV, 40 mA). In oneembodiment, the X-ray diffraction pattern includes characteristic peaksat about 12.0 to about 12.8 and about 15.4 to about 21.0.

In one embodiment, the present invention relates to a crystallineobeticholic acid Form C characterized by a Differential Scanningcalorimetry (DSC) thermogram having an endotherm value at about 98±2°C., as measured by a Mettler DSC 823e instrument. In one embodiment, theDifferential Scanning calorimetry (DSC) thermogram has an endothermvalue at about 98±2° C., as measured by a Mettler DSC 823e instrument.

In one embodiment, the present invention relates to a crystallineobeticholic acid, wherein said crystalline obeticholic acid is Form Cand has a purity greater than about 90%. In one embodiment, the purityof said crystalline obeticholic acid Form C is determined by HPLC. Inone embodiment, the present invention relates to a crystallineobeticholic acid Form C, or a pharmaceutically acceptable salt, solvateor amino acid conjugate thereof. In one embodiment, the solvate is ahydrate. In one embodiment, the purity is greater than about 92%. In oneembodiment, the purity is greater than about 94%. In one embodiment, thepurity is greater than about 96%. In one embodiment, the purity isgreater than about 98%. In one embodiment, the purity is greater thanabout 99%.

In one embodiment, the present invention relates to a crystallineobeticholic acid, wherein said crystalline obeticholic acid is Form Cand has a potency greater than about 90%. In one embodiment, the purityof said crystalline obeticholic acid Form C is determined by HPLC and/orother analytical procedures known in the art. In one embodiment, thepresent invention relates to a crystalline obeticholic acid Form C, or apharmaceutically acceptable salt, solvate or amino acid conjugatethereof. In one embodiment, the solvate is a hydrate. In one embodiment,the potency is greater than about 92%. In one embodiment, the potency isgreater than about 94%. In one embodiment, the potency is greater thanabout 96%. In one embodiment, the potency is greater than about 98%. Inone embodiment, the potency is greater than about 99%.

In one embodiment, the present invention relates to a crystallineobeticholic acid Form C that contains a total of less than about 4% ofone or more impurities selected from 6-ethylursodeoxycholic acid,3α-hydroxy-6α-ethyl-7-cheto-5β-cholan-24-oic acid,6β-ethylchenodeoxycholic acid,3α,7α-dihydroxy-6-ethyliden-5β-cholan-24-oic acid, chenodeoxycholicacid, and3α(3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oyloxy)-7α-hydroxy-6α-ethyl-5β-cholan-24-oicacid. In one embodiment, the total impurities is less than about 3.8%.In one embodiment, the total impurities is less than about 3.6%.

Example 3 of the application provides full characterization of thisnovel crystalline form of obeticholic acid.

The single crystal X-ray structure of obeticholic acid was obtained andthe absolute stereochemistry assigned. For example, the single crystalX-ray structure of crystalline obeticholic acid Form G was determinedfrom a crystal obtained from the recrystallization of obeticholic acidfrom an acetonitrile solution after cooling to 5° C. at 0.1° C./minfollowed by maturation at RT/50° C. 8 h cycles for 1 week.

The structure is orthorhombic, space group P2₁2₁2₁, and contains onemolecule of obeticholic acid in the asymmetric unit. Final R1[I>2σ(I)]=3.22%. The absolute stereochemistry of the molecule wasdetermined as shown below with a Flack parameter=−0.01 (13). Thestructure had no disorder.

A bioavailability study of obeticholic acid Form 1 (non-crystalline) vs.crystalline obeticholic acid Form F was carried out (Example 7). Theresults of the study show that that physical state of a solidobeticholic acid can play a role in the bioavailability of the moleculewhen administered orally to a subject. The plasma kinetics after oraladministration and the efficiency of the intestinal absorption and thepharmacokinetics of solid obeticholic acid Form 1 (non-crystalline) andcrystalline Form F were evaluated according to methods known in the art.Example 8 of the present invention shows the profiles of obeticholicacid plasma concentration vs time, the t_(max), C_(max) and AUC afteradministration of Form 1 or Form F of obeticholic acid (see FIGS.37-38). Crystalline Form F has a higher bioavailability than obeticholicacid Form 1 (non-crystalline). The plasma profiles show that the Form Fis absorbed more efficiently (higher AUC) and even the kinetics is moreregular, reflecting an optimal distribution of the drug in theintestinal content.

The water solubility of obeticholic acid Form 1 (non-crystalline) isslightly higher than that of Form F. Form F appears to be stable as thethermo gravimetric analysis (TGA) did not show any weight loss in thetemperature range studied.

Substantially Pure Obeticholic Acid

The present application provides substantially pure obeticholic acid andpharmaceutically acceptable salts, solvates, or amino acid conjugatesthereof:

Other names for the pharmaceutically active ingredient obeticholic acidare INT-747, 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid,6α-ethyl-chenodeoxycholic acid, 6-ethyl-CDCA, 6ECDCA, and cholan-24-oicacid, 6-ethyl-3,7-dihydroxy-,(3α,5β,6α,7α)-.

The present application provides compositions comprising obeticholicacid Form 1 and processes for the synthesis of highly pure obeticholicacid Form 1 which are safe and which produce obeticholic acid on a largescale. In one aspect, obeticholic acid Form 1 is produced on acommercial scale process. The term “commercial scale process” refers toa process which is run as a single batch of at least about 100 grams. Inone aspect, the process of the present application produces obeticholicacid Form 1 in high yield (>80%) and with limited impurities.

The term “purity” as used herein refers to the amount of obeticholicacid based on HPLC. Purity is based on the “organic” purity of thecompound. Purity does not include a measure of any amount of water,solvent, metal, inorganic salt, etc. In one aspect, the purity ofobeticholic acid is compared to the purity of the reference standard bycomparing the area under the peak. In another aspect, the known standardfor purity is an obeticholic acid reference standard. In one aspect,obeticholic acid has a purity of greater than about 96%. In one aspect,obeticholic acid has a purity of greater than about 98%. For example,the purity of obeticholic acid Form 1 is 96.0%, 96.1%, 96.2%, 96.3%,96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%,97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%,98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. For example, the purity ofobeticholic acid Form 1 is 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%,98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, or 99.9%. For example, the purity of obeticholicacid is 98.0%, 98.5%, 99.0%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. Forexample, the purity of obeticholic acid is 98.5%, 99.0%, or 99.5%. Inone embodiment, the obeticholic acid is obeticholic acid Form 1.

In one embodiment, the present invention relates to obeticholic acidhaving a purity greater than about 98%. In one embodiment, the purity isdetermined by HPLC. In another embodiment, the present invention relatesto obeticholic acid, or a pharmaceutically acceptable salt, solvate oramino acid conjugate thereof. In one embodiment, the purity is greaterthan about 98.5%. In one embodiment, the purity is greater than about99.0%. In one embodiment, the purity is greater than about 99.5%. In oneembodiment, the obeticholic acid is obeticholic acid Form 1.

The term “potency” as used herein is a measure of the amount ofobeticholic acid based on that of a known standard (e.g., acceptancecriteria of about 95% to about 102%). Potency takes into account allpossible impurities including water, solvents, organic, and inorganicimpurities. In one aspect, the known standard is obeticholic acid. Inone aspect, obeticholic acid has a potency of greater than about 96%. Inone aspect, obeticholic acid has a potency of greater than about 98%. Inone aspect, the known standard is obeticholic acid. In another aspect,potency is 100% minus the amounts of water, sulphated ash, residualsolvents, and other impurity contents such as 6-ethylursodeoxycholicacid, 3α-hydroxy-6α-ethyl-7-cheto-5β-cholan-24-oic acid,6β-ethylchenodeoxycholic acid,3α,7α-dihydroxy-6-ethyliden-5β-cholan-24-oic acid, chenodeoxycholicacid, and3α(3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oyloxy)-7α-hydroxy-6α-ethyl-5β-cholan-24-oicacid. In another embodiment, potency accounts for impurities due towater, solvent, metals, inorganic salts, and other inorganic or organicimpurities. For example, the potency of obeticholic acid Form 1 is96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%,97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%,98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%,99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.In one aspect, the potency of obeticholic acid Form 1 is 98.0%, 98.1%,98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. For example,the potency of obeticholic acid is 98.0%, 98.5%, 99.0%, 99.5%, 99.6%,99.7%, 99.8%, or 99.9%. For example, the potency of obeticholic acid is98.5%, 99.0%, or 99.5%. In one embodiment, the obeticholic acid isobeticholic acid Form 1.

In one embodiment, the present invention relates to obeticholic acidcontaining a total of less than about 2% of one or more impuritiesselected from 6-ethylursodeoxycholic acid,3α-hydroxy-6α-ethyl-7-cheto-5β-cholan-24-oic acid,6β-ethylchenodeoxycholic acid,3α,7α-dihydroxy-6-ethyliden-5β-cholan-24-oic acid, chenodeoxycholicacid, and3α(3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oyloxy)-7α-hydroxy-6α-ethyl-5β-cholan-24-oicacid. In one embodiment, the total of impurities is less than about1.5%. In one embodiment, the total of impurities is less than about1.4%. In one embodiment, the obeticholic acid is obeticholic acid Form1.

In one embodiment, obeticholic acid contains less than about 10% ofwater, less than about 9% of water, less than 8% of water, less than 7%of water, less than 6% of water, less than 5% of water, less than 4% ofwater, less than 3% of water, less than 2% of water, or less than 1% ofwater. In one embodiment, obeticholic acid contains less than about 1.2%of water. In one embodiment, obeticholic acid contains less than about1.0% of water. In one embodiment, the obeticholic acid is obeticholicacid Form 1.

In another embodiment, obeticholic acid contains not more than (NMT)0.15% of 6-ethylursodeoxycholic acid and3α,7α-dihydroxy-6-ethyliden-5β-cholan-24-oic acid. In anotherembodiment, obeticholic acid contains a total of less than about 0.07%of 6-ethylursodeoxycholic acid and3α,7α-dihydroxy-6-ethyliden-5β-cholan-24-oic acid. In one embodiment,obeticholic acid contains a total of less than about 0.06% of6-ethylursodeoxycholic acid and3α,7α-dihydroxy-6-ethyliden-5β-cholan-24-oic acid. In one embodiment,obeticholic acid contains a total of less than about 0.05% of6-ethylursodeoxycholic acid and3α,7α-dihydroxy-6-ethyliden-5β-cholan-24-oic acid. In one embodiment,the obeticholic acid is obeticholic acid Form 1.

In one embodiment, obeticholic acid contains not more than (NMT) 0.15%of 3α-hydroxy-6α-ethyl-7-cheto-5β-cholan-24-oic acid. In one embodiment,obeticholic acid contains less than about 0.07% of3α-hydroxy-6α-ethyl-7-cheto-5β-cholan-24-oic acid.

In one embodiment, obeticholic acid contains less than about 0.06% of3α-hydroxy-6α-ethyl-7-cheto-5β-cholan-24-oic acid. In one embodiment,obeticholic acid contains less than about 0.05% of3α-hydroxy-6α-ethyl-7-cheto-5β-cholan-24-oic acid. In one embodiment,the obeticholic acid is obeticholic acid Form 1.

In one embodiment, obeticholic acid contains not more than (NMT) 0.15%of 6β-ethylchenodeoxycholic acid. In one embodiment, obeticholic acidcontains less than about 0.07% of 6β-ethylchenodeoxycholic acid. In oneembodiment, obeticholic acid contains less than about 0.06% of6β-ethylchenodeoxycholic acid. In one embodiment, obeticholic acidcontains less than about 0.05% of 6β-ethylchenodeoxycholic acid. In oneembodiment, the obeticholic acid is obeticholic acid Form 1.

In one embodiment, obeticholic acid contains no more than (NMT) 3% ofchenodeoxycholic acid (CDCA). In one embodiment, obeticholic acidcontains less than about 1% of CDCA. In one embodiment, obeticholic acidcontains less than about 0.5% of CDCA. In one embodiment, obeticholicacid contains less than about 0.3% of CDCA. In one embodiment,obeticholic acid contains less than about 0.2% of CDCA. In oneembodiment, the obeticholic acid is obeticholic acid Form 1.

In one embodiment, obeticholic acid contains no more than (NMT) 4% ofCDCA and 6-ethylursodeoxycholic acid.

In one embodiment, obeticholic acid contains no more than (NMT) 1.5% of3α(3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oyloxy)-7α-hydroxy-6α-ethyl-5β-cholan-24-oicacid. In one embodiment, obeticholic acid contains less than about 1% of3α(3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oyloxy)-7α-hydroxy-6α-ethyl-5β-cholan-24-oicacid. In one embodiment, obeticholic acid contains less than about 0.07%of3α(3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oyloxy)-7α-hydroxy-6α-ethyl-5β-cholan-24-oicacid. In one embodiment, obeticholic acid contains less than about 0.06%of3α(3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oyloxy)-7α-hydroxy-6α-ethyl-5β-cholan-24-oicacid. In one embodiment, obeticholic acid contains less than about 0.05%of3α(3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oyloxy)-7α-hydroxy-6α-ethyl-5β-cholan-24-oicacid. In one embodiment, the obeticholic acid is obeticholic acid Form1.

Oral Formulation and Administration

Obeticholic acid is for oral administration. In one embodiment, theformulation is oral administration for the prevention and treatment ofFXR mediated diseases and conditions. In one embodiment, the formulationcomprises of obeticholic acid Form 1. In another embodiment, theformulation comprises of substantially pure obeticholic acid.

Formulations suitable for oral administration may be provided asdiscrete units, such as tablets, capsules, cachets (wafer capsule usedby pharmacists for presenting a drug), lozenges, each containing apredetermined amount of obeticholic acid; as powders or granules; assolutions or suspensions in aqueous or non-aqueous liquids; or asoil-in-water or water-in-oil emulsions.

Formulations of the invention may be prepared by any suitable method,typically by uniformly and intimately admixing obeticholic acid withliquids or finely divided solid carriers or both, in the requiredproportions and then, if necessary, shaping the resulting mixture intothe desired shape.

For example a tablet may be prepared by compressing an intimate mixturecomprising a powder or granules of obeticholic acid and one or moreoptional ingredients, such as a binder, lubricant, inert diluent, orsurface active dispersing agent, or by moulding an intimate mixture ofpowdered active ingredient and inert liquid diluent.

For example, one or more tablets may be administered to get to a targetdose level based on the subject's weight, e.g., a human between about 30kg to about 70 kg.

In one embodiment, the subject is a child and the formulation is used totreat biliary atresia. Biliary atresia, also known as “extrahepaticductopenia” and “progressive obliterative cholangiopathy” is acongenital or acquired disease of the liver and one of the principalforms of chronic rejection of a transplanted liver allograft. In thecongenital form, the common bile duct between the liver and the smallintestine is blocked or absent. The acquired type most often occurs inthe setting of autoimmune disease, and is one of the principal forms ofchronic rejection of a transplanted liver allograft.

Infants and children with biliary atresia have progressive cholestasiswith all the usual concomitant features: jaundice, pruritus,malabsorption with growth retardation, fat-soluble vitamin deficiencies,hyperlipidemia, and eventually cirrhosis with portal hypertension. Ifunrecognized, the condition leads to liver failure—but not kernicterus,as the liver is still able to conjugate bilirubin, and conjugatedbilirubin is unable to cross the blood-brain barrier. The cause of thecondition is unknown. The only effective treatments are certainsurgeries such as the kasai procedure, or liver transplantation

In one embodiment, the human child has had a Kasai procedure, where theKasai procedure effectively gives them a functional bile duct when theyborn either without a bile duct of its completely blocked at birth.

In addition to the ingredients specifically mentioned above, the oralformulations of the present invention may include other agents known tothose skilled in the art of pharmacy, having regard for the type offormulation in issue. Oral formulations suitable may include flavoringagents.

In one embodiment, the present invention relates to a pharmaceuticalformulation of obeticholic acid or a pharmaceutically acceptable salt,solvate, or amino acid conjugate thereof, wherein obeticholic acid isproduced by a process of the invention (obeticholic acid Form 1). Inanother embodiment, the formulation is administered orally.

In one embodiment, the formulation is in tablet form. In anotherembodiment, the formulation comprises obeticholic acid and one or morecomponents selected from microcrystalline cellulose, sodium starchglycolate, magnesium stearate, coating material, or colloidal silicondioxide. In one embodiment, the coating material is an Opadry® coatingmaterial.

In another embodiment, the formulation comprises about 0.1 mg to about1500 mg of obeticholic acid per tablet. In another embodiment, theformulation comprises about 1 mg to about 100 mg. In another embodiment,the formulation comprises about 1 mg to about 50 mg. In anotherembodiment, the formulation comprises about 1 mg to about 30 mg. Inanother embodiment, the formulation comprises about 4 mg to about 26 mg.In another embodiment, the formulation comprises about 5 mg to about 25mg. In one embodiment, the formulation comprises about 1 mg to about 2mg. In one embodiment, the formulation comprises about 1.2 mg to about1.8 mg. In one embodiment, the formulation comprises about 1.3 mg toabout 1.7 mg. In one embodiment, the formulation comprises about 1.5 mg.

In one embodiment, the formulation comprises of about 1 mg to about 25mg of obeticholic acid per tablet. In one embodiment, the formulationcomprises about 1 mg of obeticholic acid, about 180 to about 190 mg ofmicrocrystalline cellulose, about 10 to about 15 mg of sodium starchglycolate, about 1 to about 3 mg of magnesium stearate, and about 5 mgto about 10 mg of coating material. In one embodiment, the coatingmaterial is an Opadry® coating material.

In one embodiment, the formulation comprises of about 1 mg to about 25mg of obeticholic acid per tablet. In one embodiment, the formulationcomprises about 1 mg of obeticholic acid, about 185.0 mg ofmicrocrystalline cellulose, about 12.0 mg of sodium starch glycolate,about 2.0 mg of magnesium stearate, and about 8.0 mg of coatingmaterial. In one embodiment, the coating material is an Opadry® coatingmaterial.

In one embodiment, the formulation comprises of about 1 mg to about 25mg of obeticholic acid per tablet. In one embodiment, the formulationcomprises about 5 mg of obeticholic acid, about 175 to about 190 mg ofmicrocrystalline cellulose, about 10 to about 15 mg of sodium starchglycolate, about 1 to about 3 mg of magnesium stearate, and about 5 mgto about 10 mg of coating material. In one embodiment, the coatingmaterial is an Opadry® coating material.

In one embodiment, the formulation comprises of about 1 mg to about 25mg of obeticholic acid per tablet. In one embodiment, the formulationcomprises about 5 mg of obeticholic acid, about 181.0 mg ofmicrocrystalline cellulose, about 12.0 mg of sodium starch glycolate,about 2.0 mg of magnesium stearate, and about 8.0 mg of coatingmaterial. In one embodiment, the coating material is an Opadry® coatingmaterial.

In one embodiment, the formulation comprises of about 1 mg to about 25mg of obeticholic acid per tablet. In one embodiment, the formulationcomprises about 10 mg of obeticholic acid, about 170 mg to about 180 mgof microcrystalline cellulose, about 10 mg to about 15 mg of sodiumstarch glycolate, about 1 mg to about 3 mg of magnesium stearate, andabout 5 mg to about 10 mg of coating material. In one embodiment, thecoating material is an Opadry® coating material.

In one embodiment, the formulation comprises of about 1 mg to about 25mg of obeticholic acid per tablet. In one embodiment, the formulationcomprises about 10 mg of obeticholic acid, about 176.0 mg ofmicrocrystalline cellulose, about 12.0 mg of sodium starch glycolate,about 2.0 mg of magnesium stearate, and about 8.0 mg of coatingmaterial. In one embodiment, the coating material is an Opadry® coatingmaterial.

In one embodiment, the formulation comprises of about 1 mg to about 25mg of obeticholic acid per tablet. In one embodiment, the formulationcomprises about 25 mg of obeticholic acid, about 150 mg to about 160 mgof microcrystalline cellulose, about 10 mg to about 15 mg of sodiumstarch glycolate, about 1 mg to about 3 mg of magnesium stearate, about5 to about 10 mg of coating material, and about 1 to about 10 mg ofcolloidal silicon dioxide. In one embodiment, the coating material is anOpadry® coating material.

In one embodiment, the formulation comprises of about 1 mg to about 25mg of obeticholic acid per tablet. In one embodiment, the formulationcomprises about 25 mg of obeticholic acid, about 157.0 mg ofmicrocrystalline cellulose, about 12.0 mg of sodium starch glycolate,about 2.0 mg of magnesium stearate, about 8.0 mg of coating material,and about 4.0 mg of colloidal silicon dioxide. In one embodiment, thecoating material is an Opadry® coating material.

All percentages and ratios used herein, unless otherwise indicated, areby weight. The percent dimeric impurity is on an area percent basis,typically as quantified by analytical HPLC.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the inventionremains operable. Moreover, two or more steps or actions can beconducted simultaneously.

Formulation of Tablets Film Coated Tablet Quantity Reference toComponent per Tablet Function Standard 1 mg tablet Obeticholic acid 1.0mg* API HSE Microcrystalline cellulose 185.0 mg* Filler/BinderUSP-NF/EP/JP Sodium starch glycolate 12.0 mg Disintegrant USP-NF/EP/JPMagnesium stearate 2.0 mg Lubricant USP-NF/EP/JP Opadry ® II green,white, 8.0 mg Coating HSE or yellow Material Total weight 208.0 mg 5 mgtablet Obeticholic acid 5.0 mg* API HSE Microcrystalline cellulose 181.0mg* Filler/Binder USP-NF/EP/JP Sodium starch glycolate 12.0 mgDisintegrant USP-NF/EP/JP Magnesium stearate 2.0 mg LubricantUSP-NF/EP/JP Opadry ® II green, white, 8.0 mg Coating HSE or yellowMaterial Total weight 208.0 mg 10 mg tablet Obeticholic acid 10.0 mg*API HSE Microcrystalline cellulose 176.0 mg* Filler/Binder USP-NF/EP/JPSodium starch glycolate 12.0 mg Disintegrant USP-NF/EP/JP Magnesiumstearate 2.0 mg Lubricant USP-NF/EP/JP Opadry ® II green, white, 8.0 mgCoating HSE or yellow Material Total weight 208.0 mg 25 mg tabletObeticholic acid 25.0 mg* API HSE Microcrystalline cellulose 157.0 mg*Filler/Binder USP-NF/EP/JP Sodium starch glycolate 12.0 mg DisintegrantUSP-NF/EP/JP Magnesium stearate 2.0 mg Lubricant USP-NF/EP/JP Collodialsilicon dioxide 4.0 mg Glidant USP-NF/EP/JP Opadry ® II green, white,8.0 mg Coating HSE or yellow Material Total weight 208.0 mg API: Activepharmaceutical ingredient HSE = In house specification USP-NF = USPharmacopeia National Formulary Ph Eur = European Pharmacopeia JP =Japanese Pharmacopeia *obeticholic acid quantity presented assumes APIis anhydrous and 100% pure; actual amount is adjusted based on thepotency of the drug substance Lot used, and amount of microcrystallinecellulose is correspondingly decreased.

In one embodiment, the tablet comprises yellow Opadry®. In anotherembodiment, the tablet comprises white Opadry®. In another embodiment,the tablet comprises green Opadry®.

Pharmaceutical Compositions

Obeticholic acid, including obeticholic acid Form 1, substantially pureforms of obeticholic acid and crystalline forms of obeticholic acid, ora pharmaceutically acceptable salt, solvate, or amino acid conjugatethereof is useful for a variety of medicinal purposes. Obeticholic acidmay be used in methods for the prevention or treatment of FXR mediateddiseases and conditions. In one embodiment, the disease or condition isselected from biliary atresia, cholestatic liver disease, chronic liverdisease, nonalcoholic steatohepatitis (NASH), hepatitis C infection,alcoholic liver disease, primary biliary cirrhosis (PBC), liver damagedue to progressive fibrosis, liver fibrosis, and cardiovascular diseasesincluding atherosclerosis, arteriosclerosis, hypercholesteremia, andhyperlipidemia. In one embodiment, obeticholic acid Form 1 may be usedin methods for lowering triglycerides. In one embodiment, crystallineobeticholic acid may be used in methods for lowering triglycerides.Obeticholic acid Form 1 or crystalline obeticholic acid may increaseHDL. Other effects of obeticholic acid Form 1 or crystalline obeticholicacid include lowering of alkaline phosphatase (ALP), bilirubin, ALT,AST, and GGT.

In one embodiment, the present invention relates to a pharmaceuticalcomposition comprising obeticholic acid and a pharmaceuticallyacceptable carrier, wherein the obeticholic acid is produced by aprocess of the invention, e.g., obeticholic acid Form 1. In oneembodiment, the pharmaceutical composition comprises of substantiallypure obeticholic acid and a pharmaceutically acceptable carrier. Inanother embodiment, the pharmaceutical composition comprises ofcrystalline obeticholic acid and a pharmaceutically acceptable carrier.In another embodiment, the crystalline obeticholic acid is the Form C.

In one embodiment, the present invention relates to a method of treatingor preventing an FXR mediated disease or condition in a subjectcomprising administering an effective amount of obeticholic acid Form 1produced by a process of the invention or a pharmaceutical compositionthereof. In one embodiment, the present invention relates to a method oftreating or preventing an FXR mediated disease or condition in a subjectcomprising administering an effective amount of substantially pureobeticholic acid produced by a process of the invention or apharmaceutical composition thereof. In one embodiment, the presentinvention relates to a method of treating or preventing an FXR mediateddisease or condition in a subject comprising administering an effectiveamount of crystalline obeticholic acid or a pharmaceutical compositionthereof. In another embodiment, the crystalline obeticholic acid is FormC. In one embodiment, the crystalline obeticholic acid is Form A. In oneembodiment, the crystalline obeticholic acid is Form C. In oneembodiment, the crystalline obeticholic acid is Form D. In oneembodiment, the crystalline obeticholic acid is Form F. In oneembodiment, the crystalline obeticholic acid is Form G.

In another embodiment, the disease or condition is cardiovasculardisease or cholestatic liver disease and for lowering triglycerides. Inanother embodiment, the cardiovascular disease is atherosclerosis orhypercholesteremia. In another embodiment, the subject is a mammal. Inanother embodiment, the mammal is human.

In another embodiment, the compound or pharmaceutical composition isadministered orally, parenterally, or topically. In another embodiment,the compound or pharmaceutical composition is administered orally.

In one embodiment, the present invention relates to a method forinhibiting fibrosis in a subject who is suffering from a cholestaticcondition, the method comprising the step of administering to thesubject an effective amount of obeticholic acid or a pharmaceuticalcomposition thereof, wherein obeticholic acid is produced by the processof the invention. In one embodiment, the present invention relates to amethod for inhibiting fibrosis in a subject who is not suffering from acholestatic condition, the method comprising the step of administeringto the subject an effective amount of obeticholic acid or apharmaceutical composition thereof, wherein obeticholic acid is producedby the process of the invention. In embodiment, the fibrosis to beinhibited occurs in an organ where FXR is expressed.

In one embodiment, the cholestatic condition is defined as havingabnormally elevated serum levels of alkaline phosphatase, 7-glutamyltranspeptidase (GGT), and 5′ nucleotidase. In another embodiment, thecholestatic condition is further defined as presenting with at least oneclinical symptom. In another embodiment, the symptom is itching(pruritus). In another embodiment, the fibrosis is selected from thegroup consisting of liver fibrosis, kidney fibrosis, and intestinalfibrosis. In another embodiment, the cholestatic condition is selectedfrom the group consisting of primary biliary cirrhosis, primarysclerosing cholangitis, drug-induced cholestasis, hereditarycholestasis, and intrahepatic cholestasis of pregnancy. In anotherembodiment, the subject is not suffering from a cholestatic conditionassociated with a disease or condition selected from the groupconsisting of primary liver and biliary cancer, metastatic cancer,sepsis, chronic total parenteral nutrition, cystic fibrosis, andgranulomatous liver disease.

In another embodiment, the subject has liver fibrosis associated with adisease selected from the group consisting of hepatitis B; hepatitis C;parasitic liver diseases; post-transplant bacterial, viral and fungalinfections; alcoholic liver disease (ALD); non-alcoholic fatty liverdisease (NAFLD); non-alcoholic steatohepatitis (NASH); liver diseasesinduced by methotrexate, isoniazid, oxyphenistatin, methyldopa,chlorpromazine, tolbutamide, or amiodarone; autoimmune hepatitis;sarcoidosis; Wilson's disease; hemochromatosis; Gaucher's disease; typesIII, IV, VI, IX and X glycogen storage diseases; α₁-antitrypsindeficiency; Zellweger syndrome; tyrosinemia; fructosemia; galactosemia;vascular derangement associated with Budd-Chiari syndrome,veno-occlusive disease, or portal vein thrombosis; and congenitalhepatic fibrosis.

In another embodiment, the subject has intestinal fibrosis associatedwith a disease selected from the group consisting of Crohn's disease,ulcerative colitis, post-radiation colitis, and microscopic colitis.

In another embodiment, the subject has renal fibrosis associated with adisease selected from the group consisting of diabetic nephropathy,hypertensive nephrosclerosis, chronic glomerulonephritis, chronictransplant glomerulopathy, chronic interstitial nephritis, andpolycystic kidney disease.

DEFINITIONS

For convenience, certain terms used in the specification, examples andappended claims are collected here.

As used herein the term “obeticholic acid” or “OCA” refers to a compoundhaving the chemical structure:

Other chemical names for obeticholic acid include:3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid,6α-ethyl-chenodeoxycholic acid, 6-ethyl-CDCA, 6ECDCA, cholan-24-oicacid, 6-ethyl-3,7-dihydroxy-,(3α,5β, 6α,7α)- and INT-747. The CASregistry number for obeticholic acid is 459789-99-2. This term refers toall forms of obeticholic acid, e.g., non-crystalline, crystalline andsubstantially pure.

As used herein the term “crystalline obeticholic acid” refers to anycrystalline form of a compound having the chemical structure:

Crystalline obeticholic acid means that the compound is crystallizedinto a specific crystal packing arrangement in three spatial dimensionsor the compound having external face planes. The crystalline form ofobeticholic acid (or a pharmaceutically acceptable salt, amino acidconjugate, solvate thereof) can crystallize into different crystalpacking arrangements, all of which have the same elemental compositionof obeticholic acid. Different crystal forms usually have differentX-ray diffraction patterns, infrared spectral, melting points, densityhardness, crystal shape, optical and electrical properties, stabilityand solubility. Recrystallization solvent, rate of crystallization,storage temperature, and other factors may cause one crystal form todominate. Crystals of obeticholic acid can be prepared bycrystallization under different conditions, e.g., different solvents,temperatures, etc.

As used herein, the term “crystalline obeticholic acid Form C” refers toa crystalline form of obeticholic acid with an X-ray diffraction patternthat is substantially similar to that set forth in FIG. 5, e.g., thecrystalline form as characterized in Example 3.

As used herein, the term “substantially pure obeticholic acid” refers toobeticholic acid that has a potency of greater than about 95%. Thepotency of the obeticholic acid takes into account impurities includinge.g., water, solvents, and other organic and inorganic impurities thatare in a sample of obeticholic acid. In another embodiment, the knownstandard for potency is 100% obeticholic acid, and the potency isdetermined by subtracting percentages of impurities such as solvent,water, and other organic and inorganic impurities from 100% of the knownstandard. In one aspect, the inorganic impurities include e.g.,inorganic salts and sulphated ash. In one aspect, the organic impuritiesinclude 6-ethylursodeoxycholic acid,3α-hydroxy-6α-ethyl-7-cheto-5β-cholan-24-oic acid,6β-ethylchenodeoxycholic acid,3α,7α-dihydroxy-6-ethyliden-5β-cholan-24-oic acid, chenodeoxycholicacid, and3α(3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oyloxy)-7α-hydroxy-6α-ethyl-5β-cholan-24-oicacid. The amounts of the impurities can be determined by proceduresknown in the art, e.g., HPLC, NMR, or methods from US Pharmacopeial, orEuropean Pharmacopeia, or a combination of two or more of these methods.

As used herein, the term “purity” refers to a chemical analysis of acompound obtained from e.g., HPLC. In one embodiment, the purity of acompound is compared to the purity of the reference standard, e.g.,obeticholic acid, via the area under their respective peak forcomparisons. In one embodiment, purity accounts for the organicimpurities in a sample.

As used herein, the term “reaction mixture” refers a mixture of one ormore substances combined together. In one embodiment, the mixing orcombining of the substances causes a chemical transformation or changein one or more of the original substances.

As used herein, the term “obeticholic acid Form 1” refers tonon-crystalline obeticholic acid. In one embodiment, this form ofobeticholic acid is produced via a crystalline obeticholic acid as asynthetic intermediate. For example, this form of obeticholic acid isproduced by the process of the application via crystalline obeticholicacid Form C as the synthetic intermediate. In one embodiment,obeticholic acid Form 1 is the form that it used as the pharmaceuticallyactive ingredient. See Example 5 for more details.

“Treating”, includes any effect, e.g., lessening, reducing, modulating,or eliminating, that results in the improvement of the condition,disease, disorder, etc. “Treating” or “treatment” of a disease stateincludes: inhibiting the disease state, i.e., arresting the developmentof the disease state or its clinical symptoms; or relieving the diseasestate, i.e., causing temporary or permanent regression of the diseasestate or its clinical symptoms.

“Preventing” the disease state includes causing the clinical symptoms ofthe disease state not to develop in a subject that may be exposed to orpredisposed to the disease state, but does not yet experience or displaysymptoms of the disease state.

“Disease state” means any disease, disorder, condition, symptom, orindication.

The term “effective amount” as used herein refers to an amount ofobeticholic acid (e.g., an FXR-activating ligand) that produces an acuteor chronic therapeutic effect upon appropriate dose administration. Theeffect includes the prevention, correction, inhibition, or reversal ofthe symptoms, signs and underlying pathology of a disease/condition(e.g., fibrosis of the liver, kidney, or intestine) and relatedcomplications to any detectable extent.

“A therapeutically effective amount” means the amount of obeticholicacid that, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on obeticholicacid, the disease and its severity and the age, weight, etc., of themammal to be treated.

A therapeutically effective amount of obeticholic acid can be formulatedwith a pharmaceutically acceptable carrier for administration to a humanor an animal. Accordingly, obeticholic acid or its formulations can beadministered, for example, via oral, parenteral, or topical routes, toprovide an effective amount of the compound. In alternative embodiments,obeticholic acid prepared in accordance with the present invention canbe used to coat or impregnate a medical device, e.g., a stent.

“Pharmacological effect” as used herein encompasses effects produced inthe subject that achieve the intended purpose of a therapy. In oneembodiment, a pharmacological effect means that primary indications ofthe subject being treated are prevented, alleviated, or reduced. Forexample, a pharmacological effect would be one that results in theprevention, alleviation or reduction of primary indications in a treatedsubject. In another embodiment, a pharmacological effect means thatdisorders or symptoms of the primary indications of the subject beingtreated are prevented, alleviated, or reduced. For example, apharmacological effect would be one that results in the prevention orreduction of primary indications in a treated subject.

The invention also comprehends isotopically-labeled obeticholic acid, orpharmaceutically acceptable salts, solvate, or amino acid conjugatesthereof, which are identical to those recited in formulae of theinvention and following, but for the fact that one or more atoms arereplaced by an atom having an atomic mass or mass number different fromthe atomic mass or mass number most commonly found in nature. Examplesof isotopes that can be incorporated into obeticholic acid, orpharmaceutically acceptable salts, solvate, or amino acid conjugatesthereof include isotopes of hydrogen, carbon, nitrogen, fluorine, suchas ³H, ¹¹C, ¹⁴C and ¹⁸F.

Obeticholic acid, or pharmaceutically acceptable salts, solvates, oramino acid conjugates thereof that contain the aforementioned isotopesand/or other isotopes of other atoms are within the scope of the presentinvention. Isotopically-labeled obeticholic acid, or pharmaceuticallyacceptable salts, solvates, or amino acid conjugates thereof, forexample those into which radioactive isotopes such as ³H, ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes areparticularly preferred for their ease of preparation and detectability.Further, substitution with heavier isotopes such as deuterium, i.e., ²H,can afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements and, hence, may be preferred in some circumstances,isotopically labeled obeticholic acid, or pharmaceutically acceptablesalts, solvates, or amino acid conjugates thereof can generally beprepared by carrying out the procedures disclosed in the Schemes and/orin the Examples of the invention, by substituting a readily availableisotopically labeled reagent for a non-isotopically labeled reagent. Inone embodiment, obeticholic acid, or pharmaceutically acceptable salts,solvates, or amino acid conjugates thereof are not isotopicallylabelled. In one embodiment, deuterated obeticholic acid is useful forbioanalytical assays. In another embodiment, obeticholic acid, orpharmaceutically acceptable salts, solvates, or amino acid conjugatesthereof are radiolabelled.

“Geometric Isomers” means the diastereomers that owe their existence tohindered rotation about double bonds. These configurations aredifferentiated in their names by the prefixes cis and trans, or Z and E,which indicate that the groups are on the same or opposite side of thedouble bond in the molecule according to the Cahn-Ingold-Prelog rules.

“Solvates” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Obeticholicacid may have a tendency to trap a fixed molar ratio of solventmolecules in the crystalline solid state, thus forming a solvate. If thesolvent is water the solvate formed is a hydrate, when the solvent isalcohol, the solvate formed is an alcoholate. Hydrates are formed by thecombination of one or more molecules of water with one of the substancesin which the water retains its molecular state as H₂O, such combinationbeing able to form one or more hydrate. Additionally, the compounds ofthe present invention, for example, the salts of the compounds, canexist in either hydrated or unhydrated (the anhydrous) form or assolvates with other solvent molecules. Nonlimiting examples of hydratesinclude monohydrates, dihydrates, etc. Nonlimiting examples of solvatesinclude ethanol solvates, acetone solvates, etc.

“Tautomers” refers to compounds whose structures differ markedly inarrangement of atoms, but which exist in easy and rapid equilibrium. Itis to be understood that obeticholic acid may be depicted as differenttautomers. It should also be understood that when obeticholic acid andsynthetic intermediates of the invention have tautomeric forms, alltautomeric forms are intended to be within the scope of the invention,and the naming of obeticholic acid does not exclude any tautomer form.Obeticholic acid and synthetic intermediates of the invention can existin several tautomeric forms, including the keto-enol. For example, inketo-enol tautomerism a simultaneous shift of electrons and a hydrogenatom occurs. Tautomers exist as mixtures of a tautomeric set insolution. In solid form, usually one tautomer predominates. Even thoughone tautomer may be described, the present invention includes alltautomers of the present compounds.

It is to be understood accordingly that the isomers arising fromasymmetric carbon atoms (e.g., all enantiomers and diastereomers) areincluded within the scope of the invention, unless indicated otherwise.Such isomers can be obtained in substantially pure form by classicalseparation techniques and by stereochemically controlled synthesis.Furthermore, the structures and other compounds and moieties discussedin this application also include all tautomers thereof. Alkenes caninclude either the E- or Z-geometry, where appropriate. Obeticliolieacid and synthetic intermediates may exist in stereoisomeric form, andtherefore can be produced as individual stereoisomers or as mixtures.

A “pharmaceutical composition” is a formulation containing obeticholicacid in a form suitable for administration to a subject. In oneembodiment, the pharmaceutical composition is in bulk or in unit dosageform. It is can be advantageous to formulate compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active reagent calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active reagent and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active agent for the treatment of individuals.

The unit dosage form is any of a variety of forms, including, forexample, a capsule, an IV bag, a tablet, a single pump on an aerosolinhaler, or a vial. The quantity obeticholic acid (e.g., a formulationof obeticholic acid, or a pharmaceutically acceptable salt, solvate, oramino acid conjugate thereof) in a unit dose of composition is aneffective amount and is varied according to the particular treatmentinvolved. One skilled in the art will appreciate that it is sometimesnecessary to make routine variations to the dosage depending on the ageand condition of the patient. The dosage will also depend on the routeof administration. A variety of routes are contemplated, including oral,pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous,intramuscular, intraperitoneal, inhalational, buccal, sublingual,intrapleural, intrathecal, intranasal, and the like. Dosage forms forthe topical or transdermal administration of a compound of thisinvention include powders, sprays, ointments, pastes, creams, lotions,gels, solutions, patches and inhalants. In one embodiment, obeticholicacid is mixed under sterile conditions with a pharmaceuticallyacceptable carrier, and with any preservatives, buffers, or propellantsthat are required.

The term “flash dose” refers to obeticholic acid formulations that arerapidly dispersing dosage forms.

The term “immediate release” is defined as a release of obeticholic acidfrom a dosage form in a relatively brief period of time, generally up toabout 60 minutes. The term “modified release” is defined to includedelayed release, extended release, and pulsed release. The term “pulsedrelease” is defined as a series of releases of drug from a dosage form.The term “sustained release” or “extended release” is defined ascontinuous release of obeticholic acid from a dosage form over aprolonged period.

A “subject” includes mammals, e.g., humans, companion animals (e.g.,dogs, cats, birds, and the like), farm animals (e.g., cows, sheep, pigs,horses, fowl, and the like) and laboratory animals (e.g., rats, mice,guinea pigs, birds, and the like). In one embodiment, the subject ishuman. In one embodiment, the subject is human child (e.g., betweenabout 30 kg to about 70 kg). In one embodiment, the human child has hada Kasai procedure, where the Kasai procedure effectively gives them afunctional bile duct when they born either without a bile duct of itscompletely blocked at birth.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

While it is possible to administer obeticholic acid directly without anyformulation, obeticholic acid is usually administered in the form ofpharmaceutical formulations comprising a pharmaceutically acceptableexcipient and obeticholic acid. These formulations can be administeredby a variety of routes including oral, buccal, rectal, intranasal,transdermal, subcutaneous, intravenous, intramuscular, and intranasal.Oral formulation of obeticholic acid are described further herein underthe section entitled “Oral Formulation and Administration”.

In one embodiment, obeticholic acid can be administered transdermally.In order to administer transdermally, a transdermal delivery device(“patch”) is needed. Such transdermal patches may be used to providecontinuous or discontinuous infusion of a compound of the presentinvention in controlled amounts. The construction and use of transdermalpatches for the delivery of pharmaceutical agents is well known in theart. See, e.g., U.S. Pat. No. 5,023,252. Such patches may be constructedfor continuous, pulsatile, or on demand delivery of pharmaceuticalagents.

In one embodiment of the present invention, there is provided apharmaceutical formulation comprising at least obeticholic acid asdescribed above in a formulation adapted for buccal and/or sublingual,or nasal administration. This embodiment provides administration ofobeticholic acid in a manner that avoids gastric complications, such asfirst pass metabolism by the gastric system and/or through the liver.This administration route may also reduce adsorption times, providingmore rapid onset of therapeutic benefit. The compounds of the presentinvention may provide particularly favorable solubility profiles tofacilitate sublingual/buccal formulations. Such formulations typicallyrequire relatively high concentrations of active ingredients to deliversufficient amounts of active ingredients to the limited surface area ofthe sublingual/buccal mucosa for the relatively short durations theformulation is in contact with the surface area, to allow the absorptionof the active ingredient. Thus, the very high activity of obeticholicacid, combined with its high solubility, facilitates its suitability forsublingual/buccal formulation.

Obeticholic acid is preferably formulated in a unit dosage form, eachdosage containing from about 0.1 mg to about 1500 mg. In anotherembodiment, the formulation comprises about 1 mg to about 100 mg. Inanother embodiment, the formulation comprises about 1 mg to about 50 mg.In another embodiment, the formulation comprises about 1 mg to about 30mg. In another embodiment, the formulation comprises about 4 mg to about26 mg. In another embodiment, the formulation comprises about 5 mg toabout 25 mg. In one embodiment, the formulation comprises about 1 mg toabout 2 mg. In one embodiment, the formulation comprises about 1.2 mg toabout 1.8 mg. In one embodiment, the formulation comprises about 1.3 mgto about 1.7 mg. In one embodiment, the formulation comprises about 1.5mg. The term “unit dosage form” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient as described above.

Obeticholic acid is generally effective over a wide dosage range. Forexamples, dosages per day normally fall within the range of about 0.0001to about 30 mg/kg of body weight. In the treatment of adult humans, therange of about 0.1 to about 15 mg/kg/day, in single or divided dose, isespecially preferred. In embodiment, the formulation comprises about 0.1mg to about 1500 mg. In another embodiment, the formulation comprisesabout 1 mg to about 100 mg. In another embodiment, the formulationcomprises about 1 mg to about 50 mg. In another embodiment, theformulation comprises about 1 mg to about 30 mg. In another embodiment,the formulation comprises about 4 mg to about 26 mg. In anotherembodiment, the formulation comprises about 5 mg to about 25 mg. In oneembodiment, the formulation comprises about 1 mg to about 2 mg. In oneembodiment, the formulation comprises about 1.2 mg to about 1.8 mg. Inone embodiment, the formulation comprises about 1.3 mg to about 1.7 mg.In one embodiment, the formulation comprises about 1.5 mg. However, itwill be understood that the amount of obeticholic acid actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the form of obeticholic acidadministered, the age, weight, and response of the individual patient,and the severity of the patient's symptoms, and therefore the abovedosage ranges are not intended to limit the scope of the invention inany way. In some instances dosage levels below the lower limit of theaforesaid range may be more than adequate, while in other cases stilllarger doses may be employed without causing any harmful side effect,provided that such larger doses are first divided into several smallerdoses for administration throughout the day.

“Process of the invention” refers to a method for preparing obeticholicacid as described herein, wherein the method comprises of crystallineobeticholic acid.

“Fibrosis” refers to a condition involving the development of excessivefibrous connective tissue, e.g., scar tissue, in a tissue or organ. Suchgeneration of scar tissue may occur in response to infection,inflammation, or injury of the organ due to a disease, trauma, chemicaltoxicity, and so on. Fibrosis may develop in a variety of differenttissues and organs, including the liver, kidney, intestine, lung, heart,etc.

The term “inhibiting” or “inhibition,” as used herein, refers to anydetectable positive effect on the development or progression of adisease or condition. Such a positive effect may include the delay orprevention of the onset of at least one symptom or sign of the diseaseor condition, alleviation or reversal of the symptom(s) or sign(s), andslowing or prevention of the further worsening of the symptom(s) orsign(s).

As used herein, a “cholestatic condition” refers to any disease orcondition in which bile excretion from the liver is impaired or blocked,which can occur either in the liver or in the bile ducts. Intrahepaticcholestasis and extrahepatic cholestasis are the two types ofcholestatic conditions. Intrahepatic cholestasis (which occurs insidethe liver) is most commonly seen in primary biliary cirrhosis, primarysclerosing cholangitis, sepsis (generalized infection), acute alcoholichepatitis, drug toxicity, total parenteral nutrition (being fedintravenously), malignancy, cystic fibrosis, and pregnancy. Extrahepaticcholestasis (which occurs outside the liver) can be caused by bile ducttumors, strictures, cysts, diverticula, stone formation in the commonbile duct, pancreatitis, pancreatic tumor or pseudocyst, and compressionduc to a mass or tumor in a nearby organ.

Clinical symptoms and signs of a cholestatic condition include: itching(pruritus), fatigue, jaundiced skin or eyes, inability to digest certainfoods, nausea, vomiting, pale stools, dark urine, and right upperquadrant abdominal pain. A patient with a cholestatic condition can bediagnosed and followed clinically based on a set of standard clinicallaboratory tests, including measurement of levels of alkalinephosphatase, γ-glutamyl transpeptidase (GGT), 5′ nucleotidase,bilirubin, bile acids, and cholesterol in a patient's blood serum.Generally, a patient is diagnosed as having a cholestatic condition ifserum levels of all three of the diagnostic markers alkalinephosphatase, GGT, and 5′ nucleotidase, are considered abnormallyelevated. The normal serum level of these markers may vary to somedegree from laboratory to laboratory and from procedure to procedure,depending on the testing protocol. Thus, a physician will be able todetermine, based on the specific laboratory and test procedure, what isan abnormally elevated blood level for each of the markers. For example,a patient suffering from a cholestatic condition generally has greaterthan about 125 IU/L alkaline phosphatase, greater than about 65 IU/LGGT, and greater than about 17 NIL 5′ nucleotidase in the blood. Becauseof the variability in the level of serum markers, a cholestaticcondition may be diagnosed on the basis of abnormal levels of thesethree markers in addition to at least one of the symptoms mentionedabove, such as itching (pruritus).

The term “organ” refers to a differentiated structure (as in a heart,lung, kidney, liver, etc.) consisting of cells and tissues andperforming some specific function in an organism. This term alsoencompasses bodily parts performing a function or cooperating in anactivity (e.g., an eye and related structures that make up the visualorgans). The term “organ” further encompasses any partial structure ofdifferentiated cells and tissues that is potentially capable ofdeveloping into a complete structure (e.g., a lobe or a section of aliver).

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

In the specification, the singular forms also include the plural, unlessthe context clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In the case of conflict, the present specificationwill control.

All percentages and ratios used herein, unless otherwise indicated, areby weight.

EXAMPLES Example 1 Synthesis of Obeticholic Acid

The compound numbers referred to in this synthetic procedure refer tothose found in Scheme 1 and the reaction that correspond to each of thesteps.

Step 1—Preparation of 3α-hydroxy-7-keto-5β-cholan-24-oic acid methylester (1)

3α-hydroxy-7-keto-5β-cholan-24-oic acid (KLCA; 500.0 g, 1.28 mol) wasesterified using methyl alcohol (2500 mL), in the presence of acidiccatalysis (sulfuric acid, 1.0 mL) and was heated up to 62° C. to 64° C.for approximately 3 hours, to yield 3α-hydroxy-7-keto-5β-cholan-24-oicacid methyl ester (1). In this reaction, the methyl alcohol acts as themethylating reagent as well as the reaction solvent. For the work-up,the pH-value was adjusted with sodium hydroxide solution (2N) to pH 7.0to 7.5. The solution was treated with activated carbon (25 g) forapproximately 30 minutes and filtered to remove the carbon solids.Alternatively, the solution was not treated with activated carbon. Toprecipitate the product, water (625 mL) at 10° C. to 15° C. was addedover 15 minutes and seeding material was added. The reaction mixture isstirred for 1 hour at 10° C. to 15° C. Another portion of water (1875mL) was added over about 20 to 25 minutes. The product suspension wasstirred for 30 minutes at 10° C. to 15° C. The product was isolated witha centrifuge and washed with a mixture of methanol and water (1:1, 350mL). The water content of the wet material was quantified by KarlFischer (KF). The material was dried in a tumble dryer under vacuum atNMT 70° C. The material can also be used in the next step withoutdrying. The yield (calculated on dried product) is 501.4 g (1.24 mol,96.8%).

Step 2—Preparation of 3α,7α-ditrimethylsilyloxy-5β-chol-6-en-24-oic acidmethyl ester (3)

Compound 1 (60.69 g, 150 mmol, calculated as dry substance), containingresidual water and methanol, was charged into the reactor under inertconditions and was dissolved in tetrahydrofuran (THF, 363 mL). Water andmethanol were removed by repeated azeotropic distillation atapproximately 65° C. and normal pressure. THF was added to the residueas necessary and the distillation was repeated approximately 4 times.The remaining solution must have a final water content of <0.05% (KarlFischer Titration). This solution was pre-cooled to −20° C. to −25° C.and then chlorotrimethylsilane (73.33 g, 675 mmol, 4.5 equivalents) wasadded in about 30 to 45 minutes. Under nitrogen atmosphere, lithiumdiisopropyl amide (28% LDA solution, 900 mmol) and THF (504 mL) werecharged to a separate inert reactor and cooled to −20° C. to −25° C. Thedry, cooled solution of compound 1, THF (84 mL), andchlorotrimethylsilane was charged into the LDA solution at −20° C. to

−25° C. Then, the reaction mixture was stirred for approximately 2hours. For the workup, the reaction mixture was added to a pre-cooledaqueous solution of citric acid (34.6 g in 300 mL) at 2° C. to 8° C.After the addition, the aqueous phase was separated and discarded. Fromthe organic phase, the liquid was removed by vacuum distillation atmaximum 50° C. The isolated residue contained compound 3 and someresidual solvents and was used ‘as is’ in the next step.

Step 3—Preparation of 3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oicacid methyl ester (4)

Compound 3 (164.68 g, 300 mmol, calculated as dried substance) solutionin THF was charged into an inert reactor. At a maximum temperature of50° C., residual amounts of THF were distilled off under vacuum. Thewater content in the residue was limited to ≦0.5% (Karl Fischertitration) in order to proceed. The residue was then dissolved indichloromethane (200 mL) and pre-cooled to −60° C. to −65° C.Acetaldehyde (33.8 mL, 600 mmol) was then added. Under nitrogenatmosphere, dichloromethane (700 mL) and boron trifluoride (16 wt %solution in acetonitrile, 318 g, 750 mmol) acetonitrile complex werecharged into a separate reactor and then cooled to −60° C. to −65° C. At−60° C. to −65° C., the dry compound 3 solution was added. The reactionmixture was stirred for approximately two hours at −60° C. to −65° C.,heated up to 23° C. to 28° C., stirred for another approximately 3 hoursand cooled to approximately 2° C. to 10° C. for the hydrolysis/work-up.For the workup, the cooled solution from the reactor was added to apre-cooled aqueous solution of 50% wt. caustic soda (40 mL) and 660 mLof water. After about 10 minutes of intensive stirring, the phases wereseparated and the (lower) organic layer was transferred to a separatereactor. From the organic layer, the solvent was removed by distillationat NMT 50° C. as far as possible. The residue, consisting of compound 4and some remaining acetonitrile and dichloromethane, was discharged intodrums. Compound 4A, a mixture of E/Z-isomers can also be prepared by theprocedure described above for Step 3.

Step 4—Preparation of 3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oicacid (5)

Compound 4 (258.37 g, 600 mmol, calculated as dried substance) wascharged into an inert reactor. At a temperature of NMT 50° C., residualamounts of solvent were distilled off under vacuum. The residue wasdissolved in methanol (360 mL) and water (54 mL) and caustic soda 50%wt. (54 mL) were added. The reaction mixture was heated up to 49° C. to53° C. and stirred at this temperature for at least 2 hours. The pH ofthe reaction mixture is checked and verified to be >12. If the pH is<12, additional NaOH is added and the 2 hour reaction time is repeated.The solution was diluted with water (1000 mL) and the temperature wasadjusted to 25° C. to 35° C. For the workup, reaction mixture wasallowed to rest for at least 30 minutes. The phases were separated andthe lower aqueous layer was transferred into a separate reactor and theorganic layer was discarded. Ethyl acetate (1400 mL) and aqueous citricacid (244 g in 480 mL) were added with intensive stirring to the aqueouslayer. The reaction mixture was stirred at 25° C. to 35° C. for 10minutes. The phases were separated and the lower aqueous layer wasdiscarded. Ethyl acetate was distilled off from the organic layer andreplaced with ethyl acetate (800 mL). This operation was repeated untilthe water content of the distillate was NMT 1% or until a constantboiling point was reached. The suspension was cooled to 20° C. to 25°C., stirred for 30 minutes, and then the product was isolated and washedwith ethyl acetate (100 mL, 3 to 4 times). Drying was done in a tumbledryer under vacuum at approximately 60° C. The yield is 118.71 g (47.5%from KLCA) of crude compound 5. Compound 4A, a mixture of E/Z isomersalso can be used as starting material to produce compound 5A, a mixtureof E/Z isomers.

Crude compound 5 was then crystallized using ethanol. The crude compoundfor crystallization can also be a mixture of E/Z isomers, compound 5A.Ethanol (390 to 520 mL) and crude compound 5 (130 g) were charged intoan inert reactor. To dissolve the crude compound 5, the reaction mixturewas heated to reflux. Then, the reaction mixture was cooled in acontrolled cooling ramp to 15° C. to 20° C. within 3 to 5 hours by alinear profile. The crystalline compound 5A was isolated using acentrifuge and then washed with ethyl acetate (50-100 mL, 2 times).Drying was done in the tumble dryer under vacuum and at approximately60° C. This leads to 85.8 g (66%) yield. A sample was taken to measureassay, purity, and moisture of the purified compound 5. Purifiedcompound 5 is the E isomer of3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid. See example 2 forfull details regarding the identification and characterization ofpurified compound 5. Isolation of the purified compound 5, the E isomer,can be optional. The E isomer and Z isomers have different solubilities.The E isomer is less soluable and crystallizes such that the Z isomercan be washed away.

An alternative method to prepare compound 5 is as follows. Compound 4(111.96 g) was charged into the inert reactor. At maximum 50° C.residual amounts of solvent (e.g., acetonitrile, dichloromethane) weredistilled off under vacuum. The residue was dissolved in methanol (156mL) and cooled to about 10° C. Tap-water (23.4 mL) and caustic soda 50%(23.4 mL) were added. The reaction mixture was stirred for about fourhours at about 20° C. to about 25° C. The solution was diluted withtap-water (433 mL) and toluene (144 mL) was added. After stirring, thephases were separated and the lower, aqueous layer was transferred intothe inert reactor. The organic layer was discarded. Acetic acidethylester (607 mL) and a solution of citric acid (105.7 g in 208 mL ofwater) were added during intensive stirring to the aqueous layer. Thephases were separated and the lower, aqueous layer was discarded. Theorganic layer was transferred into the inert reactor. From the organiclayer acetic acid ethylester was distilled off and replaced with aceticacid ethylester (347 mL). In one embodiment, this operation was repeatedwith acetic acid ethylester (173 mL) until the water content of thedistillate was not more than about 1% or until a constant boiling pointwas reached. The present suspension was cooled to 20° C. to 25° C.Compound 5 was isolated and washed with acetic acid ethylester (3 to 4times each 43 mL) with inert centrifuge. Drying was done in the tumbledryer under vacuum and approximately 60° C. (64.8% yield based oncompound 1). Compound 4A (a mixture of E/Z isomers) can also be used asstarting material for Step 4 to produce Compound 5A (a mixture of E/Zisomers).

Step 5—Preparation of 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid(6)

A mixture of purified compound 5 (110 g, 264 mmol), water (1100 mL),caustic soda solution (35.8 mL, 682 mmol) at 50% and palladium catalyst(Pd/C, 11 g) were charged to a hydrogenation reactor. The temperaturewas adjusted to 25° C. to 35° C. and the reactor was flushed three timeswith nitrogen (2 bar) and three times with hydrogen (1 bar). Thesepressure values were given relative to ambient pressure (=0 bar). Ahydrogen pressure of 5 bar was applied and the reaction mixture washeated up to 100° C. (for isomerisation to the alpha position) over aperiod of 1.5 hours and then stirred for 3 hours while maintaining thehydrogen pressure at 4.5 to 5 bar. The reaction mixture is then cooledto 40° C. to 50° C. For the workup, the Pd/C is filtered off. To thefiltrate, n-butyl acetate (1320 mL) and hydrochloric acid (67.8 mL, 815mmol, 37%) were added. The aqueous phase was separated and discarded.The organic phase was treated with activated carbon (5.5 g) for about 10minutes at 40 to 50° C. The activated carbon was filtered off and thefiltrate was condensed by distillation and the resulting suspension wascooled to 15° C. to 20° C. within 2 to 3 hours. The precipitatedcompound 6 was isolated and washed with n-butyl acetate (160 mL). Theproduct was filtered using a pressure filter. Drying was done in thepressure filter under vacuum at approximately 60° C. This leads to 89.8g (81.2%) of Compound 6. Compound 5A, a mixture of E/Z isomers, can beused in step 5 to prepare Compound 6.

Step 6—Preparation of 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid(obeticholic acid)

A mixture of compound 6 (86 g, 205.4 mmol), water (688 mL) and 50%sodium hydroxide solution (56.4 mL) was reacted with sodium borohydride(7.77 g, 205.4 mmol) in a mixture of 50% wt. sodium hydroxide solution(1.5 mL) and water (20 mL) at 90° C. to 105° C. The reaction mixture washeated to reflux and stirred for at least 3 hours. For the workup, afterthe reaction was complete, the reaction mixture was cooled toapproximately 80° C. and transferred to a cooled reactor. At 30° C. to50° C., n-butyl acetate (860 mL) and citric acid (320.2 g, anhydrous) inwater (491 mL) were added. The aqueous phase was separated and discardedafter checking the pH-value to make sure that it was acidic. The organicphase was transferred and distilled. The residue is diluted with n-butylacetate and was slowly cooled to 15° C. to 20° C. and the crudeobeticholic acid was filtered using a centrifuge. The wet product wascrystallized from n-butyl acetate. The product obeticholic acid wasisolated and washed with n-butyl acetate (43 mL, 4 times) in an inertpressure filter. Drying was done in the pressure filter under vacuum atapproximately 80° C. This led to 67.34 g (77.9%) of crystallineobeticholic acid. See example 3 for full details regarding theidentification and characterization of crystalline obeticholic acid.

Step 7—Preparation of obeticholic acid Form 1

Crystalline obeticholic acid Form C (58 g) was dissolved in water (870mL) and caustic soda solution (50%, 8.7 mL, 166 mmol) at 30° C. to 40°C. The mixture was stirred until all solid has dissolved. The productwas precipitated using the following workup. The obeticholic acidsolution was slowly added via a filter to diluted hydrochloric acid(37%, 16.05 mL, 193 mmol) in water (870 mL) at 30° C. to 40° C. Thesuspension was stirred for approximately 30 minutes at 30° C. to 40° C.and then cooled to not more than (NMT) 20° C. The product was isolatedand washed with water (465 mL, 6 times) in the inert pressure filter.Drying was done in the pressure filter under vacuum at a temperature ofNMT 50° C. This led to 53.2 g (91.7%) of obeticholic acid Form 1.

Example 2 Characterization ofE-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid (5)

Compound 5 is the key intermediate for the process of the application.The compound was isolated from ethyl acetate and was then crystallizedfrom ethanol. The highly pure compound 5 allows for efficient and highyielding production of compound 6 and subsequently crystallineobeticholic acid Form C and obeticholic acid Form 1, includingsubstantially pure obeticholic acid.

The structure of compound 5 from step 4 in example 1 was confirmed using¹H NMR, ¹³C NMR, and mass spec. Crude product from step 4 resulted in amajor product at retention time (RT) 27.457 min and a minor product atRT 28.078 min in the UV chromatogram generated by quality control method1 by means of LC/MS-coupling. The two products are the E/Z isomers ofcompound 5:

These two isomers show the same accurate mass and the same fragmentationin the MS/MS spectrum. They cannot be distinguished by the massspectrometric data.

Using a semi-preparative method to isolate the E/Z isomer peaks, thestructures of the E/Z isomers were confirmed using a two stage approach.The HPLC quality control method 1 used a non-volatile phosphoric acidbuffer and thus, direct LC/MS coupling with the non-volatile buffer wasnot possible. Preliminary tests for adjustment of the method showed thatonly a UPLC method allowed for very high plate numbers for adequateseparation of the E/Z isomers. The two stage approach was the following:Step A was identification of the E/Z isomers in two samples with the newdeveloped UPLC/MS method and Step B was isolation of the fraction of theE/Z isomer peaks with the HPLC method 2 and subsequent identificationwith the UPLC/MS method 1. The experimental details of the methods wereas follows:

TABLE C 1. MS compatible UPLC method (method 1) Instrument: Accela UPLCcoupling with LTQ FTSpectrometer (ThermoScientific) Column: 200 × 2 mmHypersil Gold 1.9 μm Eluent: A: Water + 10 mM Ammoniumformiat + 0.1%Formic acid B: Acetonitrile Gradient: 45% B in 20 minutes to 60% B (10min isocratic) Flow: 0.4 ml/min, 40° C. column temperature Detection:MS: ESI positive and negative ions; UV: PDA 200.600 nm Mass resolution:R = 100000 ICR Sample: 1 mg/ml in water/acetonitrile (1:1), 3 μl/20 μlinjected 2. HPLC (method 2) Instrument: Agilent1100 HPLC (AgilentTechnologies) Column: 125 × 4 mm Purospher STAR C18 5 Lm Eluent: A:Water pH 2.6 with phosphoric acid B: Acetonitrile Gradient: 30% B in 10minutes to 35% B in 30 minutes to 60% B In 1 minutes to 90% B (9 minisocratic) Flow: 1 ml/min, 35° C. column temperature Detection: UV: DAD200-400 nm (UVA 200 nm) Sample: 10 mg/ml in water/acetonitrile (9:1), 25μl injected

The results are shown in FIGS. 1 and 2. FIGS. 1 and 2 are UPLC UV/MSchromatograms for “crude compound 5” (FIG. 1) and compound 5 “purifiedreference” (FIG. 2) obtained on a high performance UPLC column. For FIG.1, the sample was dissolved at 1 mg/mL in ACN/H₂O 1:1; 200×2 mm HypersilGOLD R122; LMA:H20+10 mM AF+0.1% HFo; LMB:ACN; 45%-20-60% (10); 0.4mL/min; 40° C.; UVA=200 nm; 3 μL injection volume. For FIG. 2, thesample was dissolved at 1 mg/ml in ACN/H₂O; 200×2 mm Hypersil GOLD R122;A: 10 mM AF+0.1% HFo; B:ACN; 45%-60% B (10); 0.4 mL/min; 204 injectionvolume. In both samples, the molecular weight of the main component (RT9.92 min) and of the minor component (RT 10.77 min) was the same asexpected and the accurate masses of the two compounds were consistentwith the structures provided as shown in Tables D and E of data of thepositive and negative ion measurement show below:

TABLE D Data of the positive ion measurement RT (min) Ion m/z FormulaStructure proposal 9.98 417.30008 C₂₆H₄₁O₄ M + H E Isomer ΔM 0.35 ppm833.59381 C₅₂H₈₁O₈ 2M + H E Isomer ΔM 1.45 ppm 850.61938 C₅₂H₈₄O₈N 2M +NH4 E Isomer ΔM 0.28 ppm 10.77 417.30023 C₂₆H₄₁O₄ M + H Z Isomer833.59409 C₅₂H₈₁O₈ 2M + H Z Isomer 850.61984 C₅₂H₈₄O₈N 2M + NH4 Z Isomer

TABLE E Data of the negative ion measurement RT(min) Ion m/z FormulaStructure proposal 9.98 415.28520 C₂₆ H₃₉ O₄ M − H Z Isomer ΔM −0.44 ppm461.29051 C₂₇ H₄₁ O₆ M + Formiat Z Isomer ΔM −0.76 ppm 831.57683 C₅₂ H₇₉O₈ 2M − H Z Isomer ΔM −1.46 ppm 10.77 415.28545 C₂₆ H₃₉ O₄ M − H EIsomer 461.29069 C₂₇ H₄₁ O₆ M + Formiat E Isomer 831.57739 C₅₂ H₇₉ O₈ 2M− H E Isomer

To ensure the portability of the quality control HPLC method 2, theoriginal separation was repeated exactly under the prescribedconditions. The main peak and the minor peak were isolated assemipreparative. The resulting UV chromatogram with the marked positionsof the trapped fractions is shown in FIG. 3. FIG. 3 is a UV chromatogramof crude compound 5 using HPLC method 2; 125×4 mm Purospher STAR C18 5μm AG; LMA:H2O pH 2.6mit H₃PO₄; LMB:ACN; 30% B-10-35%-30-60%-1-90% (9);1 mL/min; 35° C.; UVA-200 nm; ohne MS; 25 mL. Subsequently, the isolatedfractions were separately analyzed with the newly developed UPLC/MSmethod. For the evaluation of the accurate ion trace of thequasimolecular ion [2M+NH4] at 850.61914±3 ppm was used. The resultingchromatograms of the main peak fraction, the minor peak fraction and ofthe two samples are shown in FIG. 4 (A-D). The MS studies showed thatthe two peaks generated by quality control method 2 at RT 27.457 min andat RT 28.078 min are two isomers with the formula C₂₆H₄₀O₄. This formulais consistent with the structure proposed for the E/Z isomers. Thus, thedevelopment of the UPLC-MS method has shown that the E/Z isomers of3α-hydroxy-ethyliden-7-keto-5β-cholic-24 acid are chromatographicallyseparable with high resolution. The accurate MS data from the FR-ICRmass spectrometer are consistent with the structure proposed for the E/Zisomers. For both isomers, the same formula C₂₆H₄₀O₄ was derived.

Due to the semi-preparative isolation of the E/Z-isomer peaks with HPLCmethod 2 and subsequent identification with the UPLC-MS method we canshow that the two peaks generated by the quality control method 2 (RT27.457 minutes and RT 28.078 minutes, see FIG. 1) are the two isomerswith the formula C₂₆H₄₀O₄. This formula is consistent with the structureproposal of the E/Z-isomers. In conjunction with the NMR resultsdescribed below the following assignments were obtained: RT 27.457minutes belongs to the E-isomer and RT 28.078 minutes to the Z-isomer.

The assignment of the ¹H and ¹³C shifts for the E isomer of3α-hydroxy-ethyliden-7-keto-5β-cholic-24 acid are shown below. Shiftswere estimated according to “L. Bettarello et al., II Farmaco 55 (2000),51-55 (substance 3α-hydroxy-7-keto-5β-cholan-24-oic acid).

TABLE F

¹H Shift Assignment (¹H-NMR, 500 MHz, 303K, CD₃OD) Chemical shift [ppm]Intensity [H] Multiplicity Assignment 6.10 1 Q 25 3.61 1 M 3 2.69 1 DD 52.28 2 DT 23 1.72 3 D 26 1.05 3 S 19 0.99 3 D 21 0.70 3 S 18

TABLE G ¹³C Shift Assignment (¹³C-NMR, 125 MHz, 303K, CD₃OD) Chemicalshift [ppm] Multiplicity Assignment 207.5 S  7 178.1 S 24 145.3 S  6130.4 D 25 71.0 D  3 56.0 S 17 52.0 and 50.1 D each  8 and 14 46.9 D  544.7 S 13 40.7 D  9 40.3 T  12* 38.3 T  4* 36.5 D 20 35.8 S 10 35.4 T  132.3 and 32.0 T each 22 and 23 30.5 T  2* 29.4 T  16* 27.0 T  15* 23.2 Q19 22.4 T 11 18.9 Q 21 12.7 Q 26 12.5 Q 18 S = singlet D = doublet T =triplet Q = quartet M = multiplet DD = doublet of doublets DT = doubletof triplets

Example 3 Characterization of Crystalline Obeticholic Acid Form C

Full solid-state characterization of the product from step 6 of Scheme 1and Example 1 showed that the obeticholic acid is crystalline. Thiscrystalline form is labeled Form C. Below is a table that summarizes thecharacterization of crystalline obeticholic acid Form C:

TABLE G Summary of Crystalline Obeticholic Acid Form C CharacteristicsTechnique Crystalline Obeticholic Acid Form C appearance White powderNMR Consistent with supplied structure ca. 3.5% w/w heptane XRPDCrystalline TGA Weight losses between r.t. to 85° C. (0.4%) and 85-115°C. (4.1%) DSC Endotherm with onset of 97.9° C. GVS Slightly hygroscopic,1.2% water uptake at 90% RH Karl Fisher Water Determination 1.5% w/wStability at 40° C./75% RH No change in form or crystallinity

Thermal Analysis

DSC (Differential Scanning calorimetry) data were collected on a MettlerDSC 823e equipped with a 34 position auto-sampler. The instrument wascalibrated for energy and temperature using certified indium. Typically0.5-1 mg of each sample, in a pin-holed aluminium pan, was heated at 10°C.·min⁻¹ from 25° C. to 350° C. A nitrogen purge at 50 ml·min⁻¹ wasmaintained over the sample. The instrument control and data analysissoftware was STARe v 9.20.

TGA (Thermo-Gravimetric Analysis) data were collected on a MettlerTGA/SDTA 851e equipped with a 34 position auto-sampler. The instrumentwas temperature calibrated using certified indium. Typically 5-10 mg ofeach sample was loaded onto a pre-weighed aluminium crucible and washeated at 10° C.·min⁻¹ from ambient temperature to 300° C. A nitrogenpurge at 50 was maintained over the sample. The instrument control anddata analysis software was STARe v 9.20.

Two weight loss steps were observed by TGA of crystalline obeticholicacid Form

C. The first took place between room temperature (r.t.) and 85° C.(0.41%) and the second occurred between 85° C.-115° C. (4.10%). Thefirst weight loss step can be attributed to water loss with the secondstep being attributed to the loss of the remaining water (waterresponsible for around 1.2% weight loss) and the loss of bound heptane(ca. 3.4% weight loss). Crystalline obeticholic acid Form C containedbetween 0.15 and 0.2 moles solvent (heptane) and ca. 1.5% w/w (0.3moles). The DSC thermogram of crystalline obeticholic acid Form Ccontained one endotherm. This was fairly sharp and had an onset ofaround 98° C. See FIG. 6. Different solvents would have differentboiling points and therefore would evaporate at different temperatureswithin the DSC and TGA experiments.

X-Ray Powder Diffraction (XRPD) Analysis Bruker AXS C2 GADDS

X-Ray Powder Diffraction patterns were collected on a Bruker AXS C2GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA), automated XYZstage, laser video microscope for auto sample positioning and a HiStar2-dimensional area detector. X-ray optics consisted of a single Gobelmultilayer mirror coupled with a pinhole collimator of 0.3 mm. A weeklyperformance check was carried out using a certified standard NIST 1976Corundum (flat plate).

The beam divergence, i.e. the effective size of the X-ray beam on thesample, was approximately 4 mm. A 0-0 continuous scan mode was employedwith a sample—detector distance of 20 cm which gives an effective 20range of 3.2°-29.7°. Typically the sample was exposed to the X-ray beamfor 120 seconds. The software used for data collection was GADDS for WNT4.1.16 and the data were analyzed and presented using Diffrac Plus EVA v9.0.0.2 or v 13.0.0.2.

Ambient conditions: Samples run under ambient conditions were preparedas flat plate specimens using powder as received without grinding.Approximately 1-2 mg of the sample was lightly pressed on a glass slideto obtain a flat surface.

Non-ambient conditions: Samples run under non-ambient conditions weremounted on a silicon wafer with heat-conducting compound. The sample wasthen heated to the appropriate temperature at ca. 10° C.·min⁻¹ andsubsequently held isothermally for ca. 1 minute before data collectionwas initiated.

Bruker AXS/Siemens D5000

X-Ray Powder Diffraction patterns were collected on a Siemens D5000diffractometer using Cu Kα radiation (40 kV, 40 mA), θ-θ goniometer,divergence of V20 and receiving slits, a graphite secondarymonochromator and a scintillation counter. The instrument is performancechecked using a certified Corundum standard (NIST 1976). The softwareused for data collection was Diffrac Plus XRD Commander v2.3.1 and thedata were analyzed and presented using Diffrac Plus EVA v 11,0.0.2 or v13.0.0.2.

Samples were run under ambient conditions as flat plate specimens usingpowder as received. Approximately 20 mg of the sample was gently packedinto a cavity cut into polished, zero-background (510) silicon wafer.The sample was rotated in its own plane during analysis. The details ofthe data collection are:

Angular range: 2 to 42 °2θ

Step size: 0.05 °2θ

Collection time: 4 s·step⁻¹

Bruker AXS D8 Advance

X-Ray Powder Diffraction patterns were collected on a Bruker D8diffractometer using Cu Kα radiation (40 kV, 40 mA), θ-2θ goniometer,and divergence of V4 and receiving slits, a Ge monochromator and aLynxeye detector. The instrument is performance checked using acertified Corundum standard (NIST 1976). The software used for datacollection was Diffrac Plus XRD Commander v 2.5.0 and the data wereanalyzed and presented using Diffrac Plus EVA v 11.0.0.2 or v 13.0.0.2.

Samples were run under ambient conditions as flat plate specimens usingpowder as received. Approximately 5 mg of the sample was gently packedinto a cavity cut into polished, zero-background (510) silicon wafer.The sample was rotated in its own plane during analysis. The details ofthe data collection are:

Angular range: 2 to 42 °2θ

Step size: 0.05 °2θ

Collection time: 0.5 s·step⁻¹

XRPD showed the powder of isolated from step 6 of the process of theinvention was collected on Bruker AXS D8 Advance. See FIG. 5. Thecorresponding data for the X-ray diffractogram is presented in the tablebelow. The software used for data collection was Diffrac Plus XRDCommander v2.6.1 and the data were analysed and presented using DiffracPlus EVA v13.0.0.2 or v15.0.0.0. Samples were run under ambientconditions as flat plate specimens using powder as received. The samplewas gently packed into a cavity cut into polished, zero-background (510)silicon wafer. The sample was rotated in its own plane during analysis.The details of the data collection are:

Angular range: 2 to 42 °2θ

Step size: 0.05 °2θ

Collection time: 0.5 s·step⁻¹

TABLE H X-ray Diffractogram Data of Crystalline Obeticholic Acid Form Cpeak Angle 2-Theta (deg) d value (Angstrom) 1 4.2 21.0203 2 6.3513.90839 3 8.298 10.64718 4 9.5 9.30229 5 11.05 8.00042 6 12.246 7.221927 12.498 7.07692 8 12.647 6.99367 9 15.497 5.71337 10 15.843 5.5895 1115.998 5.53561 12 16.346 5.41836 13 16.695 5.30601 14 16.996 5.21251 1517.849 4.96547 16 18.593 4.76844 17 18.798 4.71689 18 19.047 4.65579 1920.493 4.33041 20 20.894 4.24808

VT-XRPD (Variable Temperature-X-ray Diffraction) revealed that theendotherm seen in the DSC thermogram corresponded to the desolvation ofthe sample as no form changes were observed on heating. A temperaturedifference exists between the DSC and the VT-XRPD data as the VT-XRPDexperiment was carried out in a large space with the sample exposedwhereas the DSC experiment was carried out in a confined, closed space.This difference is around 20° C. and explains why the sample melted at amuch lower temperature in the DSC experiment and the sample stillappears crystalline at 110° C. in the VT-XRPD experiment. VT-XRPD showsthat drying of the solvent from the material resulted in loss ofcrystallinity which is consistent with the material being in a solvatedform. See FIG. 7.

Gravimetric Vapour Sorption (GVS)

Sorption isotherms were obtained using a SMS DVS Intrinsic moisturesorption analyzer, controlled by DVS Intrinsic Control software v1.0.0.30. The sample temperature was maintained at 25° C. by theinstrument controls. The humidity was controlled by mixing streams ofdry and wet nitrogen, with a total flow rate of 200 ml·min⁻¹. Therelative humidity was measured by a calibrated Rotronic probe (dynamicrange of 1.0-100% RH), located near the sample. The weight change, (massrelaxation) of the sample as a function of % RH (relative humidity) wasconstantly monitored by the microbalance (accuracy ±0.005 mg).

5 to 20 mg of sample was placed in a tared mesh stainless steel basketunder ambient conditions. The sample was loaded and unloaded at 40% RHand 25° C. (typical room conditions). A moisture sorption isotherm wasperformed as outlined below (2 scans giving 1 complete cycle). Thestandard isotherm was performed at 25° C. at 10% RH intervals over a0.5-90% RH range. Data analysis was undertaken in Microsoft Excel usingDVS Analysis Suite v6.0.0.7. Method Parameters for SMS DVS IntrinsicExperiments are as follows:

Parameters Values Adsorption - Scan 1 40-90% Desorption/Adsorption -Scan 2 90-0, 0-40% Intervals (% RH) 10 Number of Scans 2 Flow rate (ml ·min⁻¹) 200 Temperature (° C.) 25 Stability (° C. · min⁻¹) 0.2 SorptionTime (hours) 6 hour time outThe sample was recovered after completion of the isotherm andre-analyzed by XRPD.

Analysis of crystalline obeticholic acid Form C showed that the samplewas slightly hygroscopic as a mass increase of 1.18% was noted between0-90% RH. This uptake of water was steady throughout the analysis andequilibrium was reached for all steps. The hysteresis of the curve wassmall indicating that the sample readily lost the water it had taken up.XRPD analysis after the GVS analysis showed that the sample wasunchanged. See FIGS. 8A, 8B, and 8C.

Water Determination by Karl Fischer Titration (KF)

The water content of each sample was measured on a Mettler Toledo DL39Coulometer using Hydranal Coulomat AG reagent and an argon purge.Weighed solid samples were introduced into the vessel on a platinum TGApan which was connected to a subaseal to avoid water ingress. Approx 10mg of sample was used per titration and duplicate determinations weremade.

Karl Fischer analysis showed that crystalline obeticholic acid Form Ccontained 1.5% water which corresponds to about 0.3 moles water.

One Week Stability at 40° C. and 75% RH

The stability of obeticholic acid at 40° C. and 75% RH (relativehumidity) was determined as follows. A sample of obeticholic acid wasstored in a humidity chamber for one week at 40° C./75% RH. The samplewas re-analyzed by XRPD and was found to have been unchanged.

The solid state study has shown that the presence of a relatively largeamount of organic solvent is required to crystallize obeticholic acidForm C. It is highly unlikely that a sample of obeticholic acid Form 1will spontaneously crystallize to form crystalline obeticholic acid FormC on storage.

Example 4 Obeticholic Acid Tablet Formulation

The table below shows the quantitative composition of obeticholic acidtablets. The 5 mg, 10 mg, and 25 mg formulations have been used as phase3 clinical trial material.

TABLE I Film Coated Tablet Film Coated Tablet Reference to ComponentQuantity per Tablet Function Standard 1 mg tablet Obeticholic acid 1.0mg* API HSE Microcrystalline 185.0 mg* Filler/Binder USP-NF/EP/JPcellulose Sodium starch 12.0 mg Disintegrant USP-NF/EP/JP glycolateMagnesium stearate 2.0 mg Lubricant USP-NF/EP/JP Opadry ® II green 8.0mg Coating HSE or white Material Total weight 208.0 mg 5 mg tabletObeticholic acid 5.0 mg* API HSE Microcrystalline 181.0 mg*Filler/Binder USP-NF/EP/JP cellulose Sodium starch 12.0 mg DisintegrantUSP-NF/EP/JP glycolate Magnesium stearate 2.0 mg Lubricant USP-NF/EP/JPOpadry ® II green 8.0 mg Coating HSE or white Material Total weight208.0 mg 10 mg tablet Obeticholic acid 10.0 mg* API HSE Microcrystalline176.0 mg* Filler/Binder USP-NF/EP/JP cellulose Sodium starch 12.0 mgDisintegrant USP-NF/EP/JP glycolate Magnesium stearate 2.0 mg LubricantUSP-NF/EP/JP Opadry ® II green 8.0 mg Coating HSE or white MaterialTotal weight 208.0 mg 25 mg tablet Obeticholic acid 25.0 mg* API HSEMicrocrystalline 157.0 mg* Filler/Binder USP-NF/EP/JP cellulose Sodiumstarch 12.0 mg Disintegrant USP-NF/EP/JP glycolate Magnesium stearate2.0 mg Lubricant USP-NF/EP/JP Collodial silicon 4.0 mg GlidantUSP-NF/EP/JP dioxide Opadry ® II green 8.0 mg Coating HSE or whiteMaterial Total weight 208.0 mg API: Active pharmaceutical ingredient HSE= In house specification USP-NF = US Pharmacopeia National Formulary PhEur = European Pharmacopeia JP = Japanese Pharmacopeia *obeticholic acidquantity presented assumes API is anhydrous and 100% pure; actual amountis adjusted based on the potency of the drug substance Lot used, andamount of microcrystalline cellulose is correspondingly decreased.

Example 5 Characterization of Obeticholic Acid Form 1

Obeticholic acid Form 1 refers to the non-crystalline form ofobeticholic acid. This form of obeticholic acid can be produced via acrystalline obeticholic acid as a synthetic intermediate. Obeticholicacid Form 1 can be used as the pharmaceutically active ingredient.Obeticholic acid Form 1 was characterized and analyzed as follows.

Batch 1 of obeticholic acid form 1 was characterized using the followingtechniques: assessment by X-ray powder diffraction (XPRD) forcrystallinity, ¹H and ¹³C nuclear magnetic resonance (NMR), Fouriertransform infrared spectroscopy (FT-IR), optical assessment (e.g.,particle shape/size), thermal properties (e.g., differential scanningcalorimetry (DSC) and thermo-gravimetric analysis (TGA)), waterdetermination by Karl Fischer (KF), storage at 40° C. and 75% RH andreanalysis after 2 weeks by XRPD, pKa by potentiometric method, Log P/D(octanol/water) by potentiometry, and stability to moisture usinggravimetric vapour sorption (GVS; e.g., complete sorption-desorptioncycle with analysis of solid collected by XRPD). Five other batches(e.g., batch 2, 3, 4, 5, and 6) of obeticholic acid Form 1 were alsocharacterized and compared using the following techniques: assessment byXRPD and comparison to main batch 1 pattern, ¹H and ¹³C NMR, FT-IR,optical assessment (e.g., particle shape/size), thermal properties(e.g., DSC, TGA, and hot-stage microscopy), and water determination byKF.

X-Ray Powder Diffraction (XRPD) Analysis

X-Ray Powder Diffraction patterns were collected on a Bruker AXS C2GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA), automated XYZstage, laser video microscope for auto-sample positioning and a HiStar2-dimensional area detector. X-ray optics consists of a single Gobelmultilayer mirror coupled with a pinhole collimator of 0.3 mm. The beamdivergence, i.e. the effective size of the X-ray beam on the sample, wasapproximately 4 mm. A θ-θ continuous scan mode was employed with asample—detector distance of 20 cm which gives an effective 2θ range of3.2°-29.7°. Typically the sample was exposed to the X-ray beam for 120seconds. The software used for data collection was GADDS for WNT 4.1.16and the data were analyzed and presented using Diffrac Plus EVA v9.0.0.2 or v 13.0.0.2.

Samples run under ambient conditions were prepared as flat platespecimens using powder as received without grinding. Approximately 1-2mg of the sample was lightly pressed on a silicon wafer to obtain a flatsurface. The diffractograms show that obeticholic acid Form 1 isnon-crystalline (See, FIG. 10 and FIG. 11).

NMR Characterization

NMR spectra were collected on a Bruker 400 MHz instrument equipped withan auto-sampler and controlled by a DRX400 console. Automatedexperiments were acquired using ICONNMR v4.0.4 (build 1) running withTopspin v 1.3 (patch level 8) using the standard Bruker loadedexperiments. For non-routine spectroscopy, data were acquired throughthe use of Topspin alone. Samples were prepared in d6-DMSO, unlessotherwise stated. Off-line analysis was carried out using ACDSpecManager v 9.09 (build 7703).

FIG. 12 shows the ¹H NMR spectrum for batch 1. ¹H NMR spectra of batches2-6 were also recorded and compared with the spectrum of batch 1. SeeFIG. 13. The spectra are all similar, but with varying amounts of water.Some differences are noted in the integration of the large group ofprotons between 0.75 ppm and 2 ppm, where peaks overlap and cannot beintegrated separately. Table J shows the total number of protonsintegrated in the spectra of batches 1-6, taking into account thevariation in the 0.75-2 ppm region.

TABLE J Number of H by integration Batch number (excluding COOH) 1 43 242 3 40 4 41 5 42 6 41-42

The carboxylic acid proton has been excluded, so the number of protonsshould be 43, but it actually varies from 40 to 43 between the 6spectra. However, the area where the variation comes from (0.75-2 ppm)is quite wide, and due to the quality of the baseline, this

integration cannot be relied upon.

As the spectrum could not be fully assigned and the integration varied,a ¹³C NMR spectrum of batch 2 was recorded. FIG. 14 shows the DEPTQspectrum, where CH₂ and quaternary carbons peaks point up, while CH₃ andCH groups point down. There are thirteen peaks pointing down, whichcorrespond to nine CHs and four CH₃ groups. This is consistent with thestructure. The peak of the carbon of the carboxylic acid was seen at 175ppm. It has been excluded from this expanded view for clarity of thearea of interest. However, there are only eleven peaks pointing up,whereas there should be twelve, as there are ten CH₂ groups and twoquaternary carbons in the molecule (excluding the carbonyl). One carbonappears to be overlapping with another signal. Therefore, a DEPT135spectrum was collected, suppressing the quaternary carbon signals, whichcould show whether the overlapping signal is quaternary. See FIG. 15. Acomparison of the DEPT135 spectrum with the DEPTQ spectrum shows thatone peak (at 42.5 ppm) disappears. There are two quaternary carbons inthe molecule, which should correspond to two peaks disappearing.Therefore the overlapping carbon signal is a quaternary one.

Further, an experiment to determine the relaxation time of the carbonswas carried out to determine where the missing quaternary carbon signalis overlapping with another carbon signal. See FIG. 16. This ¹³Cspectrum contains peaks that were integrated. This showed that peak at32.3 ppm accounts for two carbons. See FIG. 17 for an expanded view ofthe peak at 32.3 ppm. Thus, twenty-six carbons are now accounted for byintegrations (including the carboxylic acid), which is consistent withthe structure.

FT-IR by ATR

Data were collected on a Perkin-Elmer Spectrum One fitted with aUniversal ATR sampling accessory. The data were collected and analyzedusing Spectrum v5.0.1 software. See FIG. 18.

Thermal Analysis by Differential Scanning Calorimetry (DSC) andThermo-Gravimetric Analysis (TGA)

DSC data were collected on a TA Instruments Q2000 equipped with a 50position autosampler. The instrument was calibrated for energy andtemperature calibration using certified indium. Typically 0.5-3 mg ofeach sample, in a pin-holed aluminium pan, was heated at 10° C.·min⁻¹from 25° C. to 300° C. A nitrogen purge at 50 ml·min⁻¹ was maintainedover the sample. The instrument control software was Advantage for QSeries v2.8.0.392 and Thermal Advantage v4.8.3 and the data wereanalyzed using Universal Analysis v4.3A. For modulated DSC, the samplewas prepared as before, and the pan was heated at 2° C.·min⁻¹ from 25°C. to 200° C. Modulator conditions were an amplitude of 0.20° C. and aperiodicity of 40 s. The sampling interval was 1 sec/pt.

TGA data were collected on a TA Instruments Q500 TGA, equipped with a 16position autosampler. The instrument was temperature calibrated usingcertified Alumel. Typically 5-10 mg of each sample was loaded onto apre-tared platinum crucible and aluminium DSC pan, and was heated at 10°C.·min⁻¹ from ambient temperature to 350° C. A nitrogen purge at 60 wasmaintained over the sample. The instrument control software wasAdvantage for Q Series v2.8.0.392 and Thermal Advantage v4.8.3.

Thermal analysis of batch 1 was performed by DSC and TGA. The TGA trace(see FIG. 19) shows a weight loss of 1.7% between ambient temperatureand 121° C., which is likely to be loss of water. The DSC trace (seeFIG. 19) shows a broad low temperature endotherm, probably correspondingto the loss of water, followed by a small endotherm with onset at 94° C.

This second endotherm might indicate a glass transition and was furtherinvestigated by modulated DSC (see FIG. 20). This technique enablesreversible events, such as a glass transition, to be separated fromirreversible ones, such as loss of solvent or a melt of a crystallineform. The reversible heat flow trace in modulated DSC shows the glasstransition as a step with an inflexion point (Tg) at 95° C. This is highfor a glass transition and suggests that Form 1 is stable. The smallendotherm with onset at 89° C. on the non-reversible heat flow tracecorresponds to molecular relaxation of the bulk material at the glasstransition temperature.

The DSC trace (see FIG. 19) shows decomposition starting around 220° C.,which also corresponds to the TGA trace curving down.

The TGA traces of batches 1, 2, 3, 4, 5, and 6 are of similar shape(FIG. 21). The weight losses measured between ambient and 120° C. areshown in Table K. They are consistent with the varying amounts of waterobserved by NMR. These amounts were further quantified by Karl Fischer(KF) water titration. See water determination by FK.

TABLE K Summary of TGA weight losses of received samples Batch numberWeight loss by TGA 1 1.7% 2 0.6% 3 1.2% 4 0.9% 5 1.5% 6 1.6%

FIG. 22 shows the DSC traces of the six batches for comparison. Thetraces are similar, with a broad low temperature endotherm of varyingsize, consistent with varying amounts of water, followed by a smallendotherm around the glass transition temperature as seen in section DSCand TGA. The results are summarized in Table L.

TABLE L Summary of DSC results of received samples Start Batch of decom-number 1^(st) endotherm, broad 2^(nd) endotherm, small position 1 28.3J/g, Tmax = 64° C. 1.2 J/g, Tonset = 94° C. 220° C. 2  7.4 J/g, Tmax =48° C. 1.4 J/g, Tonset = 94° C. 220° C. 3 none 2.0 J/g, Tonset = 89° C.175° C. 4 14.5 J/g, Tmax = 58° C. 1.3 J/g, Tonset = 94° C. 200° C. 512.2 J/g, Tmax = 59° C. 1.2 J/g, Tonset = 94° C. 175° C. 6 28.7 J/g,Tmax = 59° C. 1.5 J/g, Tonset = 94° C. 200° C.

Polarized Light Microscopy (PLM)

Samples were studied on a Leica LM/DM polarized light microscope with adigital video camera for image capture. A small amount of each samplewas placed on a glass slide, mounted in silicone oil and covered with aglass slip, the individual particles being separated as well aspossible. The sample was viewed with appropriate magnification andpartially polarized light, coupled to a λ false-color filter.

FIGS. 23A-23F show that batches 1, 2, 3, 4, 5, and 6 are material madeup of large hard agglomerates of small irregular particles. Batches 1,2, 3, 4, 5, and 6 all look similar. No birefringence was observed underplane polarized light, which is consistent with the material beingnon-crystalline, Particle size ranges from less than 1 μm to 3 μm. Thesmall size of these particles suggests that they have been precipitatedout very quickly.

Gravimetric Vapour Sorption (GVS)

Sorption isotherms were obtained using a SMS DVS Intrinsic moisturesorption analyzer, controlled by SMS Analysis Suite software. The sampletemperature was maintained at 25° C. by the instrument controls. Thehumidity was controlled by mixing streams of dry and wet nitrogen, witha total flow rate of 200 ml·min⁻¹. The relative humidity was measured bya calibrated Rotronic probe (dynamic range of 1.0-100% RH), located nearthe sample. The weight change, (mass relaxation) of the sample as afunction of % RH was constantly monitored by the microbalance (accuracy±0.005 mg).

Typically 5-20 mg of sample was placed in a tared mesh stainless steelbasket under ambient conditions. The sample was loaded and unloaded at40% RH and 25° C. (typical room conditions). A moisture sorptionisotherm was performed as outlined below (2 scans giving 1 completecycle). The standard isotherm was performed at 25° C. at 10% RHintervals over a 0.5-90% RH range.

TABLE M Parameters Values Adsorption - Scan 1 40-90Desorption/Adsorption - Scan 2 85-Dry, Dry-40 Intervals (% RH) 10 Numberof Scans 0 Flow rate (ml · min⁻¹) 200 Temperature (° C.) 25 Stability (°C. · min⁻¹) 0.2 Sorption Time (hours) 6 hour time out

The Gravimetric Vapour Sorption (GVS) isotherm was obtained for batch 1at 25° C. and is shown in FIG. 24. The sample appears to be moderatelyhygroscopic, with a total weight change of 3.8% from 0 to 90% relativehumidity (RH). The hysteresis (area between adsorption and desorptioncurves) is small, indicating that the solid releases quite readily thewater adsorbed. No formation of hydrate is observed. There was nosignificant weight change after the whole experiment (0.3%).

The kinetics plot of the GVS (FIG. 25) shows that the adsorption of thewater occurred mostly at very high humidities and the desorption at verylow humidities. On the adsorption phase, the sample reached equilibriumquite quickly up to 80% RH and took longer to equilibrate at 90% RH. Ondesorption, the mass stabilized at all steps.

After completion of the GVS, the sample was recovered and reanalyzed byXRPD, which showed that the material was still non-crystalline (FIG.26).

Water Determination by Karl Fischer (KF)

The water content of each sample was measured on a Mettler Toledo DL39Coulometer using Hydranal Coulomat AG reagent and an argon purge.Weighed solid samples were introduced into the vessel on a platinum TGApan, which was connected to a subaseal to avoid water ingress.Approximately 10 mg of sample was used per titration and duplicatedeterminations were made.

Titration of water by coulometric Karl Fischer gave a result of 2.4 wt %water. This is slightly higher than the weight loss observed by TGA. Itcould mean that some of the water is not released from the material onheating, but it is likely to be due to the different experimentalprocedures for these two techniques.

The water content of each batch was determined by coulometric KarlFischer. Table N shows these results and compares them with earlier KarlFischer results obtained and with the weight losses observed by TGA.Data are consistent as the trend is the same in all three analyses. TheKarl Fischer data obtained earlier show lower amounts of water than theresults obtained here. This is consistent with the material beinghygroscopic, although some samples have taken up more water than others.TGA weight loss is consistently lower than the results obtained by KarlFischer titration, which might mean that some water stays trapped in thematerial and is not released on heating but might also be due to theexperimental procedure.

TABLE N Karl Fischer (KF) results and summary of water content dataBatch number KF water content Earlier KF results TGA weight loss 1 2.4%2.1% 1.7% 2 1.9% 0.4% 0.6% 3 2.5% 1.4% 1.2% 4 2.2% 0.92%  0.9% 5 2.3%0.53%  1.5% 6 2.8% 2.1% 1.6%

pKa Determination and Prediction

pKa determination data were collected on a Sirius GlpKa instrument witha D-PAS attachment. Measurements were made at 25° C. in aqueous solutionby UV and in methanol water mixtures by potentiometry. The titrationmedia was ionic-strength adjusted (USA) with 0.15 M KCl (aq). The valuesfound in the methanol water mixtures were corrected to 0% co-solvent viaa Yasuda-Shedlovsky extrapolation. The data were refined usingRefinement Pro software v1.0. Prediction of pKa values was made usingACD pKa prediction software v9.

The pKa of obeticholic acid was measured by potentiometry using methanolas a cosolvent (FIG. 27) and extrapolated to 0% co-solvent using aYasuda-Shedlovsky extrapolation (FIG. 28). The pKa enables determinationof the proportion of the neutral and the ionized form of the compound ata given pH. FIG. 29 shows the distribution of the species depending onpH.

Log P Determination

Data were collected by potentiometric titration on a Sirius GlpKainstrument using three ratios of octanol: ionic-strength adjusted (USA)water to generate Log P, Log P_(ion), and Log D values. The data wererefined using Refinement Pro software v1.0. Prediction of Log P valueswas made using ACD v9 and Syracuse KOWWIN v1.67 software.

TABLE O Predicted and measured LogP ACD (V9) Predicted LogP 5.81Measured LogP 5.54 Measured LogPion 1.58 Measured LogD7.4 2.98

LogP was predicted using ACD software then measured by potentiometry.Three titrations were performed at three different octanol/ISA waterratios, giving the difference curve plotted in FIG. 30. The black curveis the pure aqueous pKa titration and the 3 colored curves correspond tothe three octanol/ISA water ratios. The shifts in pKa enabledetermination of LogP.

The lipophilicity curve (logD as a function of pH) is shown in FIG. 31.Log D is the distribution coefficient, representing the combinedlipophilicity of all species present at a specific pH. LogP is acompound constant, which corresponds to the partition coefficient of thepure neutral species, while LogPion is that of the pure ionized species.LogP and LogPion can be determined from the lipophilicity curve, as theintersection of the Y axis with respectively the tangent at the start ofthe pH scale (when the molecule is purely in its neutral form) and thetangent at the end of the pH scale (when the molecule is completelyionized).

Two Weeks Stability at 40° C. & 75% RH and 25° C. & 97% RH

A sample of batch 1 was stored at 40° C. and 75% relative humidity (RH)in an accelerated stability testing of the solid form. Another samplewas stored at 25° C. and 97% relative humidity to check the effect ofvery high humidity. Both samples were re-analyzed by XRPD after fivedays and after two weeks. Both samples remained non-crystalline underthe two storage conditions for up to two weeks, showing that Form 1 isstable to these conditions. See FIG. 32 and FIG. 33.

The six batches analyzed were all non-crystalline. The glass transitiontemperature was measured at 95° C. with a modulated DSC experiment. Thesix batches appeared very similar with all analytical techniques used,the only difference between them being their water content, which variedfrom 1.9% to 2.8% by Karl Fischer titration. Thermal analysis showed thevarying amount of water and indicated decomposition starting around175-220° C. Measured pKa was 4.82 and LogP is 5.54. Microscopicevaluation showed large hard agglomerates of very small irregularparticles.

Stability testing showed that the material was still non-crystallineafter two weeks under accelerated conditions (40° C./75% RH) or underhigh humidity (25° C./97% RH). Gravimetric Vapour Sorption (GVS)analysis showed the material is only moderately hygroscopic, with atotal weight gain of 3.8% from 0 to 90% relative humidity (RH). Nohydrate formation was observed under GVS. The sample re-analyzed by XRPDafter GVS was still non-crystalline. The high glass transitiontemperature and the stability testing results suggest that thenon-crystalline form is stable.

Example 6 Single Crystal X-Ray Structure and Absolute Stereochemistry

The single crystal X-ray structure of obeticholic acid was determinedfrom a crystal obtained from the recrystallization of obeticholic acidfrom an acetonitrile solution after cooling to 5° C. at 0.1° C./minfollowed by maturation at RT/50° C. 8 h cycles for 1 week (see FIG. 34).The structure is consistent with Form G and a simulated XRPD pattern hasbeen generated as a reference pattern for this material. Form G can beprepared by cooling a solution of obeticholic acid in e.g.,acetonitrile.

The structure is orthorhombic, space group P2₁2₁2₁, and contains onemolecule of obeticholic acid in the asymmetric unit. Final R1[I>2σ(I)]=3.22%. The crystal exhibited prism morphology of approximatedimensions 0.4×0.4×0.3 mm. The absolute stereochemistry of the moleculewas determined as Sat chiral centres C5, C9, C10 and C14 and Rat chiralcentres C3, C6, C7, C8, C13, C17 and C22 with a Flack parameter=−0.01(13). For the inverted structure with chiral centres C5, C9, C10 and C14in the R configuration and chiral centres C3, C6, C7, C8, C13, C17 andC22 in the S configuration, the Flack parameter=1.01(13), confirming theassignation mentioned above.

Overall, the structure had a strong data set and no disorder.

The software used to assign the stereochemistry (PLATON) determines thechiral centre (C8) as an R stereocentre, whereas ACD software (and theCahn-Ingold-Prelog) assignment for (C8) is S. However, the assignment ofthe trans ring junction for B/C ring system is absolutely defined fromthe crystal structure.

Determination of the absolute structure using Bayesian statistics onBijvoet differences, (Hooft et al., J. Appl. Cryst., (2008), 41,96-103), reveals that the probability of the absolute structure aspresented being correct is 1.000, while the probabilities of theabsolute structure being a racemic twin or false are 0.000 and 0.000respectively. The Flack equivalent and its uncertainty are calculatedthrough this program to be −0.019(17).

The structure of obeticholic acid contains one 5 membered ring and 3 sixmembered rings which are fused together. Conformational analysis on the5 membered ring (C13, C14, C15, C16 and C17)) reveals that the closestpuckering descriptor for this ring is a half-chair. Conformationalanalysis on the three 6 membered rings (C1, C2, C3, C4, C5 and C10);(C5, C6, C7, C8, C9 and C10) and (C8, C9, C11, C12 C13 and C14) revealsthat the closest puckering descriptor for these rings is a chair.

Two unique intermolecular hydrogen bonds are observed in the crystalstructure. Each molecule of obeticholic acid forms a hydrogen bond totwo different symmetry related molecules of obeticholic acid, with theoxygens, O1 and O4, acting as donors to the oxygens, O3 and O1respectively, acting as acceptors, O1-H1C—O3 [D • • • A=2.7419(12)Å] andO4-H4C—O1 [D • • • A=2.6053(13)Å (see FIG. 35). These interactionsresult in a complex 3 dimensional hydrogen bonded network. The finalFourier difference map shows maximal and minimal electron densities of0.402 and −0.176 eÅ⁻³, respectively.

An overlay of the calculated XRPD pattern for the structure with theexperimental batches shows that the crystal is consistent with the bulkand is obeticholic acid Form G (see FIG. 36).

TABLE 1 Crystal data for obeticholic acid Form G Crystallizationsolvents Acetonitrile Crystallization method Maturation at RT/50° C.Empirical formula C₂₆ H₄₄ O₄ Formula weight 420.63 Temperature 100(2) KWavelength 1.54178 Å Crystal size 0.40 × 0.40 × 0.30 mm Crystal habitColourless Prism Crystal system Orthorhombic Space group P2₁2₁2₁ Unitcell dimensions a = 8.72510(10) Å α = 90° b = 12.69860(10) Å β = 90° c =22.5408(2) Å γ = 90° Volume 2497.44(4) Å³ Z  4 Density (calculated)1.119 Mg/m³ Absorption coefficient 0.574 mm⁻¹ F(000) 928

TABLE 2 Data collection and structure refinement for obeticholic acidForm G Diffractometer SuperNova, Dual, Cu at zero, Atlas Radiationsource SuperNova (Cu) X-ray Source, CuKα Data collection method omegascans Theta range for data 9.15 to 74.49° collection Index ranges −10 ≦h ≦ 10, −15 ≦ k ≦ 15, −28 ≦ l ≦ 26 Reflections collected 50001Independent reflections 5073 [R(int) = 0.0220] Coverage of independent99.4% reflections Variation in check N/A reflections Absorptioncorrection Semi-empirical from equivalents Max. and min. transmission1.00000 and 0.78605 Structure solution direct technique Structuresolution program SHELXTL (Sheldrick, 2001) Refinement techniqueFull-matrix least-squares on F² Refinement program SHELXTL (Sheldrick,2001) Function minimized Σw(F_(o) ² − F_(c) ²)²Data/restraints/parameters 5073/0/286 Goodness-of-fit on p2 1.060^(Δ/σ)max 0.001 Final R indices 5039 data; I > 2σ(I) R1 = 0.0320, wR2 =0.0859 all data R1 = 0.0322, wR2 = 0.0861 Weighting scheme calc w =1/[σ² (F_(o) ²) + (0.0503P)² + 0.5520P] where P = (F_(o) ²+ 2F_(c) ²)/3Absolute structure −0.01(13) parameter Largest diff. peak and hole 0.402and −0.1 76 eÅ⁻³ Refinement summary of the structure is as follows:Ordered Non-H atoms, XYZ Freely refining Ordered Non-H atoms, UAnisotropic H atoms (on carbon), XYZ Idealized positions riding onattached atoms H atoms (on carbon), U Appropriate multiple of U(eq) forbonded atom H atoms (on heteroatoms), Freely refining XYZ H atoms (onheteroatoms), Isotropic U Disordered atoms, OCC No disorder Disorderedatoms, XYZ No disorder Disordered atoms, U No disorder

Example 7 Bioavailability Difference Between Obeticholic Acid Form 1(Non-Crystalline) and Crystalline (Form F) Forms

The physical state of a solid obeticholic acid can play a role in thebioavailability of the molecule when administered orally to a subject(e.g., rats). The study described below was carried out to evaluate theplasma kinetics after a single oral administration and the efficiency ofthe intestinal absorption and the pharmacokinetics of solidnon-crystalline and crystalline forms of obeticholic acid. The profilesof obeticholic acid plasma concentration vs time, the t_(max), C_(max)and AUC after administration of obeticholic acid Form 1(non-crystalline) or Form F were compared (see FIGS. 37-38)

Obeticholic acid Form 1 (non-crystalline) and Form F were administeredto rats and in each animal blood was collected at different period oftimes for at least 3 hours. Six animals were studied for each form ofobeticholic acid.

Experimental Protocol:

The test substance used was obeticholic acid Form 1 (non-crystalline)and crystalline Form F. Form F can be prepared by maturation fromacetonitrile or nitromethane. The formulation was prepared as asuspension in water at pH 4. The study model is adult male SpragueDawley rats about 225 to about 250 g (Harlan Laboratories). Six animalswere used per dosage route. The dosage is PO 20 mg/kg/5 mL. The animalswere fasted overnight before treatment with the formulation ofobeticholic acid. Oral administration was performed by gastric gavage.

On day one animals were be fitted with a cannula implanted in the leftjugular vein (SOP VIVO/SAFE), anaesthesia was obtained by Isoflurane.The experiment was started after one day of recovery from surgery. About500 μL of blood (2504 of plasma) was taken via cannula in an heparinisedsyringe (Na Heparin) and collected immediately in microtubes in anice/water bath. Within 1 hour, samples were centrifuged at 10000×g for 5minutes at 4° C. Plasma was immediately transferred in microtubes andstored at −20° C. Samples of blood were collected 30 minutes, 1 hour,1.3 hour, 2 hours, and 3 hour post-dose. Plasma samples were analyzedusing the HPLC-ES/MS/MS quantitative method. Pharmacokinetics study waspreformed using non-compartmental methods.

Results:

The mean plasma concentrations of obeticholic after 20 mg/Kg b.w oralsingle dose administration of the two solid forms are reported in FIG.37. Values are the mean of six set of experiments for each formulation.The standard deviations are reported in the graph.

After administration of the crystalline form the Cmaxis achieved after1.5 hours and the plasma obeticholic acid concentration follows aregular kinetics with one maximum value and after 3 hours the dose isalmost half of the Cmax.

The kinetics profile after the administration of obeticholic acid Form 1(non-crystalline) Form 1 is different from that of the crystalline FormF. An early plasma concentration peak is obtained after 30 minutes and asecond one after 2 hours. The variability of the data in the 6 rats isvery low and this behaviour is statistically different from that of thecrystalline form. The AUC for the three hours studied is higher for thecrystalline form. The kinetics suggest that the obeticholic acid isstill present in plasma after 3 hours. It has previously beendemonstrated that the passage of obeticholic acid through the liverproduce the hepatic metabolite tauro conjugate, which is secreted intobile and accumulate in the enterohepatic circulation. Thus, themeasurement of the tauro conjugate can be used to determine the passageof the amount of obeticholic acid through the liver. The rate of tauroconjugate formation is reported in FIG. 38, which shows that the tauroconjugate formation is faster and a higher concentration is achievedafter administration of the crystalline form.

Melting Point and Glass Transition

The melting point of obeticholic acid Form 1 (non-crystalline) Form 1and crystalline Form F were measured using a conventional method. Themelting point of Chenodeoxycholic acid and Ursodeoxycholic acid weremeasured as reference compounds. Measurements have been performed intriplicate. For the crystalline form the transition from the solid toliquid state is defined as melting temperature (T_(m)) while for thenon-crystalline form is defined as glass temperature transition (T_(g)),In the table are reported the measured values expressed in both Celsius° C. and Kelvin ° K.

TABLE 3 Melting points of obeticholic acid (Form 1 and Form F) and CDCAand UDCA Experimental data Literature data Compound T_(m) (° C.) T_(g)(° C.) T_(m) (° C.) T_(g) (° C.) CDCA 136-140 — 119  98 143 163 UDCA203-207 — 203 105 Obeticholic 120-124 108-112 — — acid 235-237Experimental data Literature data T_(m) T_(g)/T_(m) T_(g)/T_(m) Compound(° K) T_(g) (° K) (° K) T_(m) (° K) T_(g) (° K) (° K) CDCA 409-413 — —392 371 0.85 416 436 UDCA 476-480 — — 477 378 0.79 Obeticholic 393-397381-385 0.75 — — 0.75 acid 508-510

Results:

The values obtained for CDCA and UDCA agree with those previouslyreported, where the melting point of UDCA is higher than that of CDCA.The transition glass temperature Tg of Form 1 (102-112° C.) is lowerthan the melting point temperature Tm of Form F (120-124° C.). Thisobserved pattern agrees with previous reported data when the two solidstate forms are compared. Form F has an additional transition at ahigher temperature (235-237° C.).

The ratio between the highest meting point temperature and the glasstransition temperature expressed in Kelvin degree is quite similar toother drugs and other bile acids. (J. Kerc et al. Thermochim. Acta, 1995(248) 81-95).

Differential Scanning Calorimetry Analysis

Differential scanning calorimetry (DSC) analysis was carried out tobetter define the melting points and the physical state of obeticholicacid crystalline and non-crystalline forms. The instrument used was aMettler Toledo DSC model 821e. Approximately 4-5 mg of each Form 1 andForm F were submitted to analysis. The compounds were exposed to thetemperature range of 30-300° C. at 10° C./min heating rate.

FIG. 39 shows the DSC curve obtained for obeticholic acid crystallineForm F. One endothermic transition at 120.04° C. was detectedcorresponding to the melting point of the compound. This result wasconfirmed also by hot stage microscopy (HSM); in the range 30°-240° C.the solid-liquid transition observed was at 122-124° C. In the DSCtrace, the peak shape and intensity obtained for Form F are in agreementwith typical behaviour showed by crystalline forms. However, the peakwidth is rather broad; this can be due to not homogeneous crystals.Thermo gravimetric analysis (TGA) did not show any weight loss in the30-300° C. temperature range.

FIG. 40 shows the DSC curve obtained for obeticholic acidnon-crystalline Form 1. One endothermic transition at 79.95° C. wasobserved. Peak shape and intensity are in agreement with behaviourexpected for non-crystalline compounds. For these substances energyrequired for solid-liquid transition (glass transition) is less than forcrystalline compounds. The thermogram did not show any weight loss inthe 30-300° C. temperature range.

Water Solubility

The water solubility of obeticholic acid Form 1 (non-crystalline) Form 1and crystalline Form F was measured following procedures known in theart. Briefly, the solid was suspended in water at a low pH (HCl 0.1mol/L) and left to equilibrate at 25° C. for one week under slightlymixing. The saturate solution was filtered and the concentration of thecompound in solution measured by HPLC-ES-MS/MS.

Results:

Water solubility (μmol/L) Form 1 17.9 Form F 9.1Form 1 present a higher solubility 17.9 μmol/L vs. 9.1 μmol/L for FormF.

According to the bioavailability data of the obeticholic acid,crystalline Form F is higher than the obeticholic acid Form 1(non-crystalline). Despite an earlier plasma concentration peak afteradministration of the Form 1, the plasma profiles show that the Form Fis absorbed more efficiently (higher AUC) and even the kinetics is moreregular, reflecting an optimal distribution of the drug in theintestinal content. Form 1 shows this early peak then a later second onewith a Cmaxlower that than of Form F.

The water solubility of the Form 1 is higher than that of Form F. Form Fappears to be stable as the thermo gravimetric analysis (TGA) did notshow any weight loss in the temperature range studied.

According to these results, Form F when administered orally appears moreefficiently absorbed by the intestine and taken up by the liver. Therate of formation of the main hepatic metabolite tauro conjugate isalmost twice for Form F compared to Form 1, suggesting a more efficienttransport and accumulation in the enterohepatic circulation and theplasma concentration after 3 hours.

Example 8 Preparation of Radiolabelled Obeticholic Acid

Radiolabelled obeticholic acid was prepared according to the schemebelow.

NMR spectra were recorded in CDCl₃ and MeOD-d₄ solution in 5-mm o.d.tubes (Norell, Inc. 507-HP) at 30° C. and were collected on VarianVNMRS-400 at 400 MHz for ¹H. The chemical shifts (δ) are relative totetramethylsilane (TMS=0.00 ppm) and expressed in ppm. LC-MS/MS wastaken on Ion-trap Mass Spectrometer on Accela-Thermo Finnigan LCQ Fleetoperating EST (−) ionization mode. HPLC was taken on Agilent 1200 series(Column: Xterra MS C8, 250×4.6 mm, 5 μm, 40° C.) in line β-Ram. Specificactivity was taken on LSA (Liquid Scintillation Analyzer, Perkin Elmer,Tri-Carb 2900TR).

Preparation of Compound 2X

To a solution of diisopropylamine (1.59 g, 15.8 mmol) in dry THF (6.0mL) was added n-BuLi (6.30 mL, 2.5 M, 15.8 mmol) at −20° C. Afterstirring the reaction mixture for 1 h at −20° C., cooled to −78° C. andTMSC1 (1.72 g, 15.8 mmol) was added followed by compound 1X (3.00 g,6.29 mmol) in dry THF (6.0 mL). The reaction mixture was stirred for 1 hat −78° C., quenched by addition of NaHCO₃ and stirred for 30 min atroom temperature. The organic layer was separated and concentrated invacuo to give the compound 2X (3.29 g, 95%) and used for next stepwithout further purification.

Preparation of Compound 3X

The [1-¹⁴C]actaldehyde (330 mCi, 5.63 mmol) (prepared from [¹⁴C]BaCO₃,SA=58.6 mCi/mmol) in toluene (1.0 mL) and acetaldehyde (130 mg, 2.95mmol) in DCM (2.0 mL) were mixed at −78° C. and then transferred to asolution of compound 2X (3.29 g, 6.00 mmol) in DCM (13.0 mL) followed byaddition of BF₃.OEt₂ (1.05 g, 7.40 mmol) at −78° C. After stirring for 1h at −78° C., the reaction mixture was allowed to warm up to 35° C. andstirred for 1 h at the above temperature. The reaction was quenched byaddition of water (10 mL), the aqueous layer was extracted with DCM, thecombined organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated in vacuo The residue was purified by column chromatographyon SiO₂ (Hexane: EtOAc=5:1 to 3:1) to give the compound 3X (102 mCi,31%, SAW 37.0 mCi/mmol) as a white solid.

¹H-NMR (CDCl₃, Varian, 400 MHz): 8 0.65 (3H, s); 0.93 (3H, d, J=6.0 Hz),1.01 (3H, s), 1.06-1.49 (12H, m), 1.62-2.04 (7H, m), 1.69 (3H, d, J=6.8Hz), 2.18-2.28 (2H, m), 2.32-2.43 (2H, m), 2.58 (1H, dd, J=12.8, 4.0Hz), 3.62-3.70 (1H, m), 3.67 (3H, s), 6.18 (1H, q, J=6.8 Hz).

Preparation of Compound 4X

To a solution of compound 3X (102 mCi, 2.75 mmol) in MeOH (6.0 mL) wasadded NaOH (220 mg, 5.50 mmol) in H₂O (3.0 mL) at room temperature.After stirring the reaction mixture for 1 h at 45° C., cooled to roomtemperature, MeOH was removed under reduced pressure and diluted withH₂O (12 mL). The aqueous layer was acidified with H₃PO₄, extracted withDCM and the organic layer was concentrated in vacuo. The residue wassuspended in Et₂O and the precipitate was collected by filtration togive the compound 4X (86.3 mCi, 85%) as a white solid.

¹H-NMR (CDCl₃, Varian, 400 MHz); 8 0.63 (3H, s), 0.92 (3H, d, J=6.0 Hz),0.99 (3H, s), 1.04-1.50 (13H, m), 1.61-2.01 (7H, m), 1.67 (3H, d, J=7.2Hz), 2.21-2.28 (2H, m), 2.35-2.41 (2H, m), 2.56 (1H, dd, J=12.8, 4.0Hz), 3.58-3.69 (1H, m), 6.16 (1H, q, J=7.2 Hz).

Preparation of Compound 5X

The mixture of compound 4X (86.3 mCi, 2.35 mmol) and 5%-Pd/C (100 mg) inaq. 0.5 M NaOH (10 mL, 5.0 mmol) was stirred for 10 h at roomtemperature under H₂ atmosphere (balloon) and then stirred for 14 h at100° C. The catalyst was removed by filtration, washed with water andthe filtrate was acidified with H₃PO₄. The precipitates was collected byfiltration, the solid was dissolved in EtOAc, washed with brine,filtered through a short pad of SiO₂ and concentrated in vacuo. Theresidual solid was recrystallization with EtOAc to give the compound 5X(67.7 mCi, 78%) as a white solid.

¹H-NMR (MeOD-d₄, Varian, 400 MHz): 8 0.71 (311, s), 0.75-0.84 (1H, m),0.81 (3H, t, J=7.4 Hz), 0.92-1.01 (1H, m), 0.96 (3H, d, J=6.4 Hz),1.06-1.38 (7H, m), 1.25 (3H, s), 1.41-1.96 (1211, m), 2.01-2.05 (1H, m),2.11-2.24 (2H, m), 2.30-2.37 (1H, m), 2.50 (1H, t, J=11.4 Hz), 2.80-2.85(1H, m), 3.42-3.49 (1H, m).

Preparation of [ethyl-1-¹⁴C]Obeticholic Acid

To a solution of compound 5X (67.7 mCi, 1.83 mmol) in aq. 2 M NaOH (4.50mL, 9.00 mmol) was added a solution of NaBH₄ (416 mg, 11.0 mmol) in 1120(2.0 ml) at 80° C. After stirring the reaction mixture for 2 h at 100°C., water (6.0 mL) was added at room temperature and acidified withH₃PO₄. The aqueous layer was extracted with DCM, dried over anhydrousNa₂SO₄, filtered through a short pad of SiO₂ and concentrated in vacuo,The residue was purified by column chromatography on SiO₂(Hexane:EtOAc=1:1 to 1:3) to give the product (44.0 mCi, 65%) as a whitesolid. The product (44.0 mCi, 1.19 mmol) and obeticholic acid (120 mg,0.285 mmol) were dissolved in EtOAc (4 mL), the solution was stirred for2 h at 50° C. and then concentrated in vacuo. The residual oil suspendedin Et₂O, the precipitate was collected by filtration to give the[ethyl-1-¹⁴C]obeticholic acid (560 mg, 38.5 mCi, SA=29 mCi/mmol) as awhite solid.

¹H-NMR (CDCl₃, Varian, 400 MHz): 8 0.66 (3H, s), 0.88 (311, s), 0.93(3H, t, J=7.2 Hz), 0.93 (3H, d, I=6.4 Hz), 0.96-1.04 (1H, m), 1.08-1.52(14H, m), 1.51-1.60 (1011, m), 2.22-2.30 (111, m), 2.36-2.44 (1H, m),3.38-3.45 (111, m), 3.71 (1H, s).

LC-MS/MS (MS: LCQ Fleet): MS Calcd.: 421.56; MS Found: 421.07 [M-H]⁻.

Radio TLC: TLC plate of silica 60 F₂₅₄, and mobile phase is EtOAc.Radiochemical purity is 98.90%, Rf=0.675

HPLC (Agilent 1200 series): Mobile phase; acetonitrile: 5 mM Phosphatebuffer (pH=3):MeOH=450:450:100. Radiochemical purity is 98.19% (β-ram),Rt=20.00 min.

[Ethyl-1-¹⁴C]obeticholic acid has a molecular formula of ¹⁴C₁C₂₅H₄₄O₄and a molecular weight of 421.46 at the specific activity of 29 mCi/mmolby LSC.

We claim:
 1. A crystalline obeticholic acid Form C characterized by anX-ray diffraction pattern including characteristic peaks at about 4.2,6.4, 9.5, 12.5, and 16.7 degrees 2-Theta.
 2. The crystalline obeticholicacid Form C according to claim 1 characterized by an X-ray diffractionpattern substantially similar to that set forth in FIG.
 5. 3. Thecrystalline obeticholic acid Form C according to claim 1, furthercharacterized by a Differential Scanning calorimetry (DSC) thermogramhaving an endotherm value at about 98±2° C.
 4. A process for preparingobeticholic acid Form 1, comprising the step of converting crystallineobeticholic acid to obeticholic acid Form
 1. 5. The process according toclaim 4, comprising the steps of reacting3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ to formcrystalline obeticholic acid, and converting crystalline obeticholicacid to obeticholic acid Form
 1. 6. The process according to claim 5,comprising the steps of reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid,reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ toform crystalline obeticholic acid, and converting crystallineobeticholic acid to obeticholic acid Form
 1. 7. The process according toclaim 6, comprising the steps of reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl esterwith NaOH to form E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid, reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid,reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ toform crystalline obeticholic acid, and converting crystallineobeticholic acid to obeticholic acid Form
 1. 8. The process according toclaim 7, comprising the steps of reacting3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester withCH₃CHO to form E- or E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oicacid methyl ester, reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl esterwith NaOH to form E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid, reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid,reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ toform crystalline obeticholic acid, and converting crystallineobeticholic acid to obeticholic acid Form
 1. 9. The process according toclaim 8, comprising the steps of reacting3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester withLi[N(CH(CH₃)₂)₂] and Si(CH₃)₃Cl to form3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester, reacting3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester withCH₃CHO to form E- or E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oicacid methyl ester, reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl esterwith NaOH to form E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid, reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid,reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ toform crystalline obeticholic acid, and converting crystallineobeticholic acid to obeticholic acid Form
 1. 10. The process accordingto claim 9, comprising the steps of reacting3α-hydroxy-7-keto-5β-cholan-24-oic acid with CH₃OH and H₂SO₄ to form3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester, reacting3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester withLi[N(CH(CH₃)₂)₂] and Si(CH₃)₃Cl to form3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester, reacting3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester withCH₃CHO to form E- or E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oicacid methyl ester, reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl esterwith NaOH to form E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid, reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid,reacting 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ toform crystalline obeticholic acid, and converting crystallineobeticholic acid to obeticholic acid Form
 1. 11. The process accordingto claim 10, wherein converting crystalline obeticholic acid Form C toobeticholic acid Form 1 comprises the step of dissolving crystallineobeticholic acid Form C in aqueous NaOH solution and adding HCl.
 12. Theprocess according to claim 10, wherein reacting3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid with NaBH₄ to formcrystalline obeticholic acid is carried out at a temperature at about85° C. to about 110° C. in a basic aqueous solution.
 13. The processaccording to claim 10, wherein reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid with Pd/C andhydrogen gas to form 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oic acid iscarried out at a temperature at about 90° C. to about 110° C. and at apressure at about 4 to about 5 bars.
 14. The process according to claim10, wherein reacting E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid methyl esterwith NaOH to form E- orE/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oic acid is carried outat a temperature at about 45° C. to about 60° C.
 15. The processaccording to claim 10, wherein reacting3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester withCH₃CHO to form E- or E/Z-3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oicacid methyl ester is carried out in a polar aprotic solvent at atemperature at about −50° C. to about −70° C. in the presence of BF₃.16. The process according to claim 10, wherein reacting3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester withLi[N(CH(CH₃)₂)₂] and Si(CH₃)₃Cl to form3α,7-ditrimethylsilyloxy-5β-chol-6-en-24-oic acid methyl ester iscarried out in a polar aprotic solvent at a temperature at about −10° C.to about −30° C.
 17. The process according to claim 10, wherein reacting3α-hydroxy-7-keto-5β-cholan-24-oic acid with CH₃OH and H₂SO₄ to form3α-hydroxy-7-keto-5β-cholan-24-oic acid methyl ester is heated for about3 hours and the pH of the reaction mixture is adjusted with an aqueousbasic solution to a pH-value of about 6.5 to about 8.0.
 18. Anobeticholic acid, or a pharmaceutically acceptable salt, solvate oramino acid conjugate thereof, having a potency of greater than about98%, greater than about 98.5%, greater than about 99.0%, or greater thanabout 99.5%.
 19. A pharmaceutical composition comprising obeticholicacid Form 1 produced by the process of claim 4 and a pharmaceuticallyacceptable carrier.
 20. A method of treating or preventing an FXRmediated disease or condition in a subject comprise of administering aneffective amount of obeticholic acid Form 1 produced by the process ofclaim 4, wherein the disease or condition is selected from biliaryatresia, cholestatic liver disease, biliary atresia, chronic liverdisease, nonalcoholic steatohepatitis (NASH), hepatitis C infection,alcoholic liver disease, primary biliary cirrhosis (PBC), liver damagedue to progressive fibrosis, liver fibrosis, and cardiovascular diseasesincluding atherosclerosis, arteriosclerosis, hypercholesteremia, andhyperlipidemia.